How to collect plasma/serum samples?
Serum: Collect whole blood samples using serum separation tubes containing a clot activator. Allow the samples to clot at room temperature for 60 minutes (note: do not shake the tubes). Centrifuge at 3000 rpm for 10 minutes at 4°C, then aliquot 200 μl of the supernatant (serum) into pre-labeled 2 ml centrifuge tubes. Snap freeze the samples in liquid nitrogen for 5-10 minutes, and store them at -80°C. Plasma: Collect whole blood samples using blood collection tubes containing an anticoagulant (EDTA or heparin sodium). Gently invert the tubes to mix immediately. Centrifuge at 3000 rpm for 10 minutes at 4°C, then aliquot 200 μl of the supernatant (plasma) into pre-labeled 2 ml centrifuge tubes. Snap freeze the samples in liquid nitrogen for 5-10 minutes, and store them at -80°C. (Note: heparin sodium anticoagulant should not be used for microbiome studies.)
How to collect Adherent Cells?
Remove the culture medium and quickly wash the cells 2-3 times with pre-cooled PBS solution. Add an appropriate amount of pre-cooled PBS solution to the culture flask and gently scrape the cells off the flask wall into the PBS solution using a cell scraper. Transfer the mixture to an appropriate centrifuge tube and centrifuge at 300g-500g for 5 minutes at 4°C. Discard the supernatant and wash the cell pellet again with pre-cooled PBS solution. Centrifuge at 1000g for 5 minutes at 4°C and discard the supernatant. Resuspend the cell pellet in pre-cooled PBS solution and measure the cell suspension concentration. Transfer a cell suspension containing 10^7 cells into a 2 mL sterile centrifuge tube, centrifuge at 1000g for 10 minutes at 4°C, and discard the supernatant. Collect the cell pellet, snap freeze it in liquid nitrogen for 5-10 minutes, and store it at -80°C.
How to collect Suspended Cells?
Measure the concentration of the cell culture. Transfer a cell suspension containing 1*10^7 cells into a 2 ml centrifuge tube. Centrifuge at 1000g for 10 minutes at 4°C, and discard the supernatant. Quickly wash the cell pellet with pre-cooled PBS (4°C) 2-3 times, each time centrifuging at 1000g for 10 minutes at 4°C and discarding the supernatant. Collect the cell pellet, snap freeze it in liquid nitrogen for 5-10 minutes, and store it at -80°C.
How to collect organoid samples?
Collecting organoid samples requires a delicate approach to preserve their structure and function. Begin by preparing your tools: sterile dishes or tubes for collection, as well as lysis buffers or preservation solutions. Carefully detach the organoids from their growth substrate, which may involve using a gentle enzyme treatment (like dispase) or mechanical scraping, depending on the type of organoid. After detachment, transfer the organoids to a pre-chilled tube containing a lysis buffer suitable for your analysis. If the organoids need to be stored, flash-freezing them in liquid nitrogen is ideal to prevent degradation. Make sure to keep the samples at -80°C until you're ready to analyze them.
How to collect baterial samples?
Measure the concentration of the microbial culture. Transfer the culture containing the required amount of microorganisms into an appropriate centrifuge tube. Centrifuge at 4000 rpm for 10 minutes at 4°C, and discard the supernatant. Wash the pellet twice with pre-cooled PBS (4°C), each time centrifuging at 1000 rpm for 10 minutes at 4°C and discarding the supernatant. Collect the microbial pellet, snap freeze it in liquid nitrogen for 5-10 minutes, and store it at -80°C. (For bacteria, it is recommended to sample during the logarithmic growth phase.)
How to collect culture supernatant?
Collecting culture supernatant is a straightforward process that requires sterile techniques. First, grow your cells in appropriate culture media until they reach the desired confluence or density. Once ready, carefully remove the culture media using a sterile pipette, ensuring not to disturb the cell layer too much. For adherent cultures, rinse gently with PBS to remove any remaining media, then add fresh media back to the cells for further incubation, if needed. After allowing enough time for the cells to produce secreted metabolites, centrifuge the media at low speed (around 300 g) to remove any cell debris. Transfer the supernatant to a clean tube without disturbing the pellet. For metabolomic analysis, filter it through a 0.22-micron filter to remove any remaining particulates. Store the supernatant at -80°C until analysis to maintain metabolite integrity.
How to collect stool samples?
Collecting stool samples requires careful handling to avoid contamination and ensure sample integrity. Provide participants with a sterile collection kit, which should include a clean container and, ideally, a scoop for easy transfer. Instruct them to collect a representative portion of the stool sample, avoiding any contact with urine or other contaminants. Once collected, the sample should be transferred into the provided container as soon as possible. For metabolomic analysis, freezing the sample at -80°C immediately is ideal to preserve the microbial and metabolite profiles. If immediate freezing isn't feasible, keeping the sample on ice until it can be processed is crucial to minimize degradation and changes in composition.
How to determine the number of biological replicates?
Determining the number of biological replicates in a study is essential for ensuring the reliability and validity of results. Generally, it depends on the expected variability in your measurements and the statistical power required to detect significant differences. A common starting point is to aim for at least three biological replicates per experimental condition, as this often provides a good balance between statistical power and resource constraints. For medical research, tipically at least 6 duplicates are required to get more reliable results.
How to normalize the sample amount for metabolomics?
Normalizing sample amounts for metabolomics is critical for accurate comparisons across samples. One common approach is to use total protein content as a normalization factor. This involves quantifying the protein concentration in each sample, using methods like the BCA assay or Bradford assay, and then adjusting the volume of each sample based on its protein concentration to ensure equal amounts are analyzed. Another strategy is to normalize based on cell count, tissue weight or liquid volume, particularly in studies where these metrics are relevant.
How to collect bacteriological solution sample for metabolomics?
To collect a bacteriological solution sample for metabolomics, begin with aseptic techniques to ensure the integrity of the sample. If you are working with a liquid culture, use a sterile pipette or syringe to draw the required volume from the culture. It's crucial to minimize the exposure of the culture to the environment to prevent contamination. Once collected, transfer the sample into a sterile tube suitable for storage and analysis. If you are interested in intracellular metabolites, you may need to quickly lyse the cells before freezing the sample. For best results, immediately flash-freeze the sample in liquid nitrogen and store it at -80°C until you're ready for metabolomic analysis.
How to prepare whole blood samples?
Preparing whole blood samples starts with proper collection techniques. First, ensure you have the right anticoagulant tubes (like EDTA or citrate) to prevent clotting. Draw blood using a sterile needle and transfer it into the tube, taking care to invert the tube gently to mix the blood with the anticoagulant. It’s important to handle the blood gently to minimize hemolysis, which can interfere with metabolomic analysis. Once collected, you can either process the blood immediately or store it temporarily at 4°C. If you're processing it right away, you may need to centrifuge the sample at around 1,500–2,000 g for 10–15 minutes to separate plasma from cellular components. After centrifugation, carefully transfer the supernatant (plasma) to a clean tube and store it at -80°C for future analysis, ensuring the integrity of the metabolites is maintained.
How to prepare EVs/ exosomes?
To prepare extracellular vesicles (EVs) or exosomes, start by collecting a suitable biological fluid, such as serum, plasma, or cell culture supernatant. After collection, centrifuge the sample at low speed (around 300 g) for 10 minutes to remove cells and debris. Follow this with a higher-speed centrifugation (10,000 g for 30 minutes) to further clear the sample of larger vesicles and proteins. Next, ultrafiltration or density gradient centrifugation can be used to isolate the exosomes from the remaining supernatant. For ultrafiltration, pass the supernatant through a filter to concentrate the vesicles. If using density gradient centrifugation, layer the supernatant onto a gradient (like sucrose or iodixanol) and centrifuge at high speeds (around 100,000 g for 2 hours) to separate the exosomes. Collect the fraction containing exosomes, and store them at -80°C until analysis to preserve their lipid and protein content.
How to prepare saliva samples collected by swab?
To prepare saliva samples collected by swab, first ensure the swab is sterile and suitable for saliva collection. Instruct the individual to avoid eating or drinking for at least 30 minutes prior to collection for more accurate results. Collect the saliva by swabbing the inner cheek, gums, or tongue for adequate sample volume. Once collected, place the swab in a sterile tube and immediately transfer it to a freezing environment, such as dry ice or a -80°C freezer.
How to prepare FFPE samples?
"Formalin-fixed paraffin-embedded (FFPE) samples require careful handling to preserve their integrity for downstream analysis. Start by ensuring the tissue is adequately fixed in formalin, typically for 24-48 hours, and then embedded in paraffin. When you’re ready to prepare the sample, cut thin sections (around 5 micrometers) using a microtome and collect them on a microscope slide or in a suitable container.
To extract the metabolites or proteins from FFPE samples, deparaffinize the sections using xylene or a xylene substitute, followed by rehydration through a series of ethanol washes. Once deparaffinized, you can use a lysis buffer to extract proteins or nucleic acids, ensuring to include protease and phosphatase inhibitors if analyzing proteins. After extraction, store the samples at -80°C until you're ready for metabolomic analysis."
How to prepare soil samples?
Preparing soil samples for metabolomic analysis involves several steps to ensure a representative sample. Start by collecting soil from various spots within the desired area to capture spatial variability. Use a clean tool to avoid contamination, and store the samples in sterile bags or containers. Aim to collect at least 100 grams of soil for a comprehensive analysis. Once collected, air-dry the soil to a consistent moisture level, as this is crucial for accurate metabolomic profiling. Grind the soil to break up clumps and achieve a homogenous sample. If needed, sieve the soil to remove larger debris. You can then extract metabolites using appropriate solvents like methanol or a water-methanol mixture. After extraction, filter the solution to remove solid particles and store it at -80°C until analysis.
How to prepare tear samples collected by strips?
To prepare tear samples collected by strips, start by ensuring that the strips are sterile and specifically designed for tear collection. Gently place the strip in the lower conjunctival sac of the eye for a few minutes to absorb tears. Be sure to instruct participants to avoid blinking excessively during this time for optimal collection. Once the strip has absorbed the tears, remove it carefully and place it into a sterile tube. If immediate analysis isn’t possible, freeze the samples at -80°C to preserve the metabolite profile. When ready for analysis, you can elute the tears from the strip into a buffer solution or solvent suitable for metabolomics, ensuring that the metabolites remain intact during this process.
How to prepare brain tissue and neuron cell samples?
"To prepare brain tissue samples for analysis, start by quickly dissecting the brain from the animal following institutional guidelines, then rinse it in ice-cold phosphate-buffered saline (PBS) to remove contaminants like blood. Dissect specific regions if needed, keeping the tissue cold throughout to preserve integrity. For studies requiring homogenized tissue (e.g., proteomics or metabolomics), place the tissue in a microcentrifuge tube with an appropriate buffer, adding protease and phosphatase inhibitors if required, and homogenize on ice. For frozen storage, flash-freeze the tissue in liquid nitrogen or on dry ice before transferring to -80°C, or, for histology, fix the tissue in paraformaldehyde or cryoprotectant before embedding in OCT or paraffin for later sectioning. These methods ensure optimal preservation for various downstream applications.
When preparing neuronal cell samples, seed primary neurons or neuronal cell lines on poly-L-lysine or laminin-coated plates and culture under optimal conditions (37°C, 5% CO₂) until they reach the required maturity. Gently wash the cells with pre-warmed PBS, then detach them with a cell scraper or enzyme-free dissociation solution, taking care to avoid excessive handling, as neurons are delicate. Collect the cell suspension and centrifuge at low speed (300-500g), resuspending in ice-cold PBS to remove residual medium. For storage, cells can be resuspended in a cryoprotectant solution (e.g., 10% DMSO in medium) and frozen gradually by initially placing in -80°C before transferring to liquid nitrogen. This approach minimizes cellular degradation, preserving sample quality for a wide range of applications."
Can we use cell samples that are preserved in DMSO (dimethyl sulfoxide) for metabolite detection?
Yes, the cell samples in DMSO would be fine for metabolite detection.
Can we do metabolite analysis on tissue samples that have been treated with RIPA lysis buffer?
If the sample has been homogenized or lysed by vibration, the release of active proteins will affect metabolites. Moreover, the high levels of salts in the samples will be precipitated during pre-processing, making it's not suitable for metabolomics analysis. If the sample was frozen directly after adding 500 ul of lysis buffer when it was collected, it is recommended to either remove the sample directly or blow dry the lysis buffer with nitrogen before performing the metabolomics analysis.
Is there any effect on metabolite detection when blood is taken from animals after anesthesia with chloral hydrate or isoflurane?
No. Blood sampling after anesthesia is suitable for metabolomics analysis.
Is it possible to send samples for widely targeted detection when magnetic beads and cells are mixed together?
Yes. The magnetic beads are coated with inorganic substances. During the centrifugation step in the pre-processing, the magnetic beads will sink to the bottom of the tube and will not get into the extraction solution.
Can we run a test using 15 ml of cell lysate? How should we concentrate the samples after receiving them?
Yes. We freeze-dry the samples directly into powder after receiving them and then add the extraction buffer.
The client wants to test lymphocyte samples, which are small in volume, but the quantity meets the requirement. Is there a problem with this?
Metabolome assay can be performed with such samples. However, during the pre-processing, we will determine whether protein quantification can be performed based on the actual cell number (visual inspection of the cell precipitation volume > 15ul) to ensure a sufficient sample volume for extraction and for metabolome detection.
Can we do non-targeted testing on root secretions? If it can be done, can the salt solution be used for detection runs? And the 0.22 μm filter membrane used by the client was a hydrophilic filter membrane. Does it affect the test?
Normally, we would concentrate the root secretion culture 30 times and then extract it with 70% methanol in water instead of directly using a salt solution for detection. Filtration by hydrophilic membranes normally does not retain small molecules that are soluble in water. ② The client can also extract the root secretion from fresh soil. The method is: add 10g of soil to 50 ml of 50% methanol in water, shake and soak, then centrifuge to get the supernatant, freeze-dry, or send it to us for freeze-drying.
What is the sample volume required for fish oil?
The main components of fish oil are lipids (such as fatty acids and glycerolipids), which are viscous, not easy to pipette directly, but can be weighed. When using fish oil for metabolite analysis, it can be referred to as tissue samples, and the sample volume should be at least 40mg (about 50uL).
What is the sample volume required when using lipid droplets for non-targeted and widely targeted detections?
Lipid droplets are important organelles that store lipids and energy in cells and can be isolated from cells or tissues. The sample volume can be referred to as organelle, with the recommended sample volume being 4×10^7 cells/0.2 g tissue (sample volume at 100% extraction efficiency). The client needs to increase the sample volume according to the efficiency of their extraction method.
How to prepare organoid samples for metabolomics analysis.
The sample volume for organoid samples can be referred: The organoids were cultured in two 24-well plates, with approximately 50 organoids per well. At the time of collection, six wells of organoids were collected and mixed into one sample to ensure that there were enough metabolites for lipid analysis.
How do we control the homogeneity of organoid sampling and testing? Do we test the concentration? Or will the samples be centrifuged and weighed the same weight?
"With respect to organoid projects, normalization can be performed using protein volumes and sample volumes with the following requirements:
① Protein volume normalization: The total amount of protein in the sample is required to be more than 10ug, which generally corresponds to 1*10E6 cells or 500mg in weight. Organoids can be digested with trypsin. Protein quantification is feasible as long as they are not agglomerated.
② Weight normalization: This method is used when the total sample volume is required to be more than 20 mg."
Can Mycobacterium tuberculosis be used for lipidomics analysis? And how to inactivate such samples?
Mycobacterium tuberculosis is sensitive to ethanol and methanol and can be inactivated with 80% methanol in water. Collect a certain amount of bacterial sediment (with a 2 ml centrifuge tube), such as 1 ml of Mycobacterium tuberculosis with OD600 of 1 (about 108), add 500 ul of 80% methanol in water, shake and mix, seal the tube with sealing film, and preserve the samples.
How should blood samples from HIV-infected patients be inactivated?
HIV-infected blood samples are inactivated with methanol (blood: methanol = 1:4). Accurately pipette a certain volume of plasma, accurately add 4 times the volume of 100% methanol, vortex for 3-5 min, and freeze the samples with liquid nitrogen, and then send them with dry ice.
How should I handle plasma versus serum for metabolomics?
When handling plasma and serum for metabolomics, it's important to recognize the differences between these two sample types and how they can influence metabolite profiles. Plasma is obtained by collecting blood with anticoagulants, preventing clotting and preserving a wider range of metabolites, including those that might be lost during clotting. Serum, on the other hand, is collected after blood has clotted, which can result in the loss of certain proteins and metabolites that may be adsorbed to the clot. We should also consider the timing of sample collection in relation to physiological changes, as metabolic profiles can vary significantly based on factors such as diet and circadian rhythms. When preparing samples for analysis, consistency in handling and processing is key, regardless of whether using plasma or serum. Properly standardizing collection, storage, and processing methods for both sample types helps ensure that the results are reliable and comparable.
Can fasting affect the metabolomic profile of blood samples?
Yes, fasting can significantly affect the metabolomic profile of blood samples. When individuals fast, their metabolism shifts to utilize stored energy sources, leading to changes in levels of various metabolites. For example, fasting may result in decreased levels of glucose and increased levels of ketone bodies as the body starts to break down fats for energy. The duration of fasting can also influence the extent of these changes. Short-term fasting might lead to modest alterations in metabolite levels, while prolonged fasting can result in more pronounced shifts in the metabolome.
Are there specific guidelines for collecting cerebrospinal fluid (CSF) for metabolomics studies?
Yes, there are specific guidelines for collecting cerebrospinal fluid (CSF) for metabolomics studies. CSF collection typically involves a lumbar puncture, and it's essential to follow sterile techniques to minimize the risk of contamination. Proper patient positioning, usually sitting or lying down, helps ensure a successful and safe procedure. Additionally, researchers should use appropriate collection tubes, often made of polypropylene to reduce the risk of adsorption of metabolites. After collection, CSF samples should be processed as quickly as possible to preserve metabolite integrity. Ideally, samples should be centrifuged to remove cells and debris, then aliquoted and frozen immediately at -80°C. This rapid processing helps maintain the stability of metabolites and reduces the risk of degradation.
Can samples be collected in the field, or do they need to be processed immediately?
Samples can be collected in the field, but immediate processing is often ideal to preserve metabolite integrity. When field collection is necessary, it’s crucial to implement proper storage and transport protocols. For instance, using portable coolers with ice packs can help keep samples at low temperatures until they can be processed. This is particularly important for biological samples, as temperature fluctuations can lead to metabolite degradation or changes due to enzymatic activity. If immediate processing isn’t feasible, it’s essential to minimize exposure to factors that might alter the metabolome, such as temperature and light. Samples should be aliquoted and frozen as quickly as possible upon returning to the lab. Implementing standard operating procedures for field sampling can help ensure the reliability of metabolomic data, allowing us to conduct studies in diverse environments without compromising sample integrity.
What are the best practices for collecting plant samples for metabolomics?
Collecting plant samples for metabolomics involves several best practices to ensure high-quality data. First, timing is essential; samples should be collected during periods of optimal growth or when specific metabolites are known to be abundant. It's also beneficial to consider environmental factors, such as temperature and humidity, which can influence metabolite levels. When collecting the samples, use clean, sterilized tools to avoid contamination. It's advisable to collect samples in the morning when metabolic processes are typically more stable. After collection, samples should be quickly frozen or dried, depending on the metabolites of interest. Snap-freezing in liquid nitrogen is an effective way to preserve the integrity of sensitive metabolites, while drying can be useful for others. Clear documentation of collection conditions and times is also critical for reproducibility.
Can repeated freeze-thaw cycles impact the metabolite content in samples?
Yes, repeated freeze-thaw cycles can significantly impact the metabolite content in samples. Each cycle can lead to the degradation of sensitive metabolites, particularly those that are unstable or prone to hydrolysis, such as certain amino acids or small organic acids. The physical stresses of freezing and thawing can also cause cellular lysis, releasing additional metabolites into the surrounding matrix and potentially skewing quantification results. To minimize these effects, we should aim to aliquot samples into smaller portions before freezing. This allows for a single thawing of each aliquot when needed, reducing the overall number of freeze-thaw cycles per sample. It's also beneficial to use cryoprotectants in some cases, which can help stabilize certain metabolites during freezing.
How to handle frozen whole blood (not plasma or serum) samples in EDTA tubes?
For the EDTA anticoagulant tubes, it can be used to take blood samples with pretreated in time (such as quenching in 50% methanol in 15 min ); if left it stand for a long time, subsequent samples will be layered with plasma on top and cells on the bottom.
As fecal samples have lots of water, how do we do sample normalization to ensure the same amount of material going into the pipeline?
If you want to solve the detection error caused by the water content of feces, you should first freeze dry the fecal samples uniformly, and then take the same amount of samples. However, due to the cycle problem, all products of stool samples are not freeze-dried temporarily (the default moisture content is the same).
Organoids are samples that cannot be weighted like tissues, and they cannot count cells. How do we normalize or organoids?
The sampling of organoids were describe follow: 1) Organoids were cultured in two 24-well plates, and each well approximately 50 organoids ;2) for sampling, one sample were collect from 6 wells and mixed into one sample to ensure sufficient metabolites for lipid analysis. Reference: Jukes Z, Freier A, Glymenaki M, Brown R, Parry L, Want E, Vorkas PA, Li JV. Lipid profiling of mouse intestinal organoids for studying APC mutations. Biosci Rep. 2021 Mar 26;41(3):BSR20202915. doi: 10.1042/BSR20202915
What is the sample requirement for yeast cells for metabolomics?
The size of one sample is about 1*10^10 yeast cells
I has samples of ground phloem into powder in liquid nitrogen for metabolomics (powder currently at -80℃). How would the thawing of samples affect the metabolite detection process.
The samples do not need to be thawed, you could directly sent tous with dry ice. If the sample freeze-thaw, the conmposition and content of metabolites will changed, since when the temperature rises, the activity of the enzyme is enhanced, which will catalyze the reaction of the metabolites.
Are we able to detect phenolic acids, flavanoids, coumarins, and lignans in the Widely-Targeted Metabolomics for Plants?
yes, In this product, we have 1100+ phenolic acids, 3100+ flavanoids, 420+ coumarins ,730+ lignans
Can we perform untargeted analysis for fungi (Calocybe indica)?
Yes, 500 mg samples are required.
For preparing cells, can we prepare more than 1 million or is the process standarized at 1.0E6?
yes, we will standarize the amout of cell by Bicinchoninic acid assay (BCA)
Do we work with non-adherent cells for human sample?
Yes, please ensure that the number of cell is more than 10^6, and we suggest you send us the cell precipitate samples.
For seed samples in plants, what is the minimum sample requirement for metabolomics analysis?
600mg seed samples will be fine for metabolomics analysis.
We have seed millet samples. What are the requirements for sample preparation? The samples are currently hulled and stored in a tube in room temperature. Do we need to ask client to dehull the seed? And what about lyophilization, frozen or samples can be shipped to us in room temperature as is.
We suggest removing the shells. And for Widely-Targeted Metabolomics for Plants, the dry seed sample can be sent at room temperature.
How to work with platelets as samples?
Yes, we have experence on that, but client should to isolate plateletsfor us. For Untargeted and TM, we need about 10^7 platelets.
How to remove the cell media completely to avoid metabolite detection in cell media but just focused on the cells. Do we have to wash it with materials like PBS for metabolomics analysis?
Yes, the client should wash the materials with appropriate volume PBS to remove the cell media.
which will be the minimum volume of a cerebral spinal fluid (CSF) for which you would do a good metabolomics coverage for disocvery (untargeted) analysis?
The minimum volume of Cerebrospinal fluid sample is 20 μL for our Untargeted Metabolomics analysis.
Do we have experiences working with mouse islet cells? (Some past experiences or publications would be great).
We have the experience of mouse islet cells for metabolomics analysis.
What are the sample requirements for Bile Acids for culture medium (human hepatocytes medium)?
We suggest 100 mg per sample (in test tube), the minimum sample size is 20mg per sample
What is the recommended and minimum sample size for embryos?
We recommend 20-30 embryos of Zebrafish.
We wants to use your Energy Metabolism Panel. Our samples are fresh tomato fruits. Is our requirement for Energy Panel also 600mg recommended, 300mg minimum?
ok. 600mg recommended, 300mg minimum sample is OK.
We want to submit endophyte samples from a plant to run Widely-Targeted Metabolomics for Plants. The samples can either be on a PDA plate or liquid culture with extract broth. What is our requirement for either of these sample types?
For solid media, please ensure scraping off the mycelium and aim for at least 100mg sample. Similarly, when dealing with lyophilized powder, collect over 100mg. With liquid medium, aim for 5ml ideally, but a minimum of 3ml is acceptable. For liquid samples, please specify the culture medium and note any concerns about substances secreted into it during the culture process.
We wants to test potato tuber tissue, callus culture, and single cell suspension.
We want to analyze and compare the metabolite changes as the samples changes format. I understand the lab procedure for tissue and cells can be different. Can we combine the data for analysis?
Yes, we can combine the data and perfom the bioinfomatic analysis.
We are interested in our past experience with mouse fecal samples for widely-targeted metabolomics. Can we provide this? -ideally they would like a full list, specifically looking at the different Glycosphingolipids we can identify, but if we can provide a summary that would be fine too
Does the client require a full list of metabolites with mouse fecal samples in the past experience for TM? Typically, we just provide a count of metabolites across various classes. -Our TM widely-targeted metabolomics can detect over 3000 compounds in mouse fecal samples, provided in an Excel format. However, if your focus is primarily on lipids like glycosphingolipids, this method might not be optimal as it's better suited for water-soluble compound detection. Instead, I recommend our quantitative lipidomics approach, boasting a database of 4000+ lipids, including 828 sphingolipids. We've detected over 1300 lipids in mouse serum samples based on past experience.
I have a client interested in a number of hormones, specifically glucocorticoids, corticosterone, cortisol, epinephrine, melatonin, and other sterioid hormones in general.
Can we detect the above compounds in our TM, untargeted, or QL? What other steroid hormones can we detect?
We're capable of detecting glucocorticoids and epinephrine. Unfortunately, we don't have the capability to detect melatonin in any biomedical samples.
What’s the sample requirement for bacterial cultures for phytohormone assay?
1. bacteria collection from solid medium
1) Sample Selection: choose 3-6 plates of bacteria from the same batch as one biological replicate
2) Sample Collection: Use a scraper or a surgical knife to scrape the bacterial biomass from the surface of the culture medium, making an effort to avoid introducing the medium. Collect a minimum of 1g for each biological replicate. 3-6 biological replicates are requested.
3) Sample Preservation: Place the collected samples in 2ml or 5ml centrifuge tubes. After collection, flash-freeze the samples in liquid nitrogen for 5 minutes and store them at -80°C.
4) Sample Shipment: Schedule dry ice shipment for the samples. Please prepare sufficient dry ice to prevent sample thawing.
How to collect bacteria samples from liquid medium.
1) Sample Selection: for the same batch of cultured samples, the culture liquid in 3 conical flasks (100ml) was selected for bacterial mixing as one biological replicate
2) Determination of Growth Phase: Assess the growth cycle by measuring the OD value of the liquid culture medium. Generally, the logarithmic growth phase is preferred, during which bacteria exhibit normal growth and accumulate a higher quantity of metabolites.
3) Sample Collection: take 45ml of culture liquid into a 50ml centrifuge tube. Centrifuge at 4000rpm at a low temperature (4°C) for 5 minutes, discard the supernatant, repeat this process 3 times to ensure a bacterial biomass of at least 1g. 3-6 biological replicates are requested.
4) Sample Washing: Gently resuspend the bacterial biomass in pre-cooled PBS and centrifuge at 4000rpm at a low temperature (4°C) for 5 minutes. Discard the supernatant and repeat this washing step 3 times to thoroughly remove the culture medium.
5) Sample Preservation and Shipment: Rapidly freeze the collected bacterial biomass in liquid nitrogen for 5 minutes and store it at -80°C. Schedule dry ice shipment for the samples, ensuring an ample supply of dry ice to prevent sample thawing.
Do you have any past experience for soil samples for Untargeted and Widely Targeted Metabolomics analysis?
We have past experience for soil samples in Untargeted and Widely Targeted Metabolomics.
What’s the process for extracting this sample type? How should the client store and ship the samples?
Sample Requirements: Each test strip sample should be immersed in tears for a minimum length of 10 mm. Clients can either cut the test strips themselves or indicate the immersed length of tears on each strip, and we will handle the cutting accordingly; Sample Extraction: Upon sample receipt, we will conduct sample extraction and analysis using well-established internal experimental protocols, with methanol as the primary extraction solvent; Sample Preservation and Shipment: Rapidly freeze the samples in liquid nitrogen and store it at -80°C. Schedule dry ice shipment for the samples, ensuring an ample supply of dry ice to prevent sample thawing.
Is tissue homogenate the same as tissues? Also for tissue samples I thought the sample recommendation is 100mg. Why is it 300mg?
100mg will be fine. it would be better to provide more than 300 mg.
We are interested in submitting supernatants from her various organoids for an untargeted metabolomics. Can you accept supernatant for untargeted metabolomics, and if so, how should I prepare the samples?
The sample preparation method for organoid supernatant can be based on the sample preparation method for cell supernatant, with the following specific steps:
Collect the supernatant in sterile tubes of appropriate size. Centrifuge at 1200 rpm for 5 minutes at 4°C to remove cell debris and impurities. Withdraw the supernatant post-centrifugation using a sterile 10 ml syringe, and pass it through a disposable needle filter (0.22μm) to eliminate any bacteria that may be present in the supernatant.
Recommended Guidelines for Samples:
Sample Size: 100 μL;
Biological Replicates: ≥ 6/group
Can we get some past experiences on human blood in Bile Acid Targeted Assay? What compounds have been detected in the past?
Our focus has primarily been on samples such as blood, tissue, and feces. In this report, we present findings from studies involving human blood, human feces, and mouse liver for your consideration.
Have we had any experience working with dates and high sugar content samples in the past, particularly using Widely Targeted Metabolomics for Plants?
We have extensive experience with dates and other high sugar content samples in plant widely targeted metabolomics assay. We provide some related project experiences, sample categories include dates, grapes and peaches.
How to send media samples to do metabolomics (any type of service) and to first lyophilize the media sample, is the media sample requirement the same?
The sample requirement is the same. 100 ul is fine.
How to provid seed materials for Amino acids Assay, how much samples will she need to provide, What are the sample requirements?
Sample type: dry samples
Recommended sample size: 600 mg;
Minimum sample size: 300 mg;
Biological Replicates: ≥ 3 group
We interested in looking at trace metals in culture supernatant. Can we detect these in our Widely Targeted Metabolomics panel or other assays?
No. that's not what our LCMS do.
We have mouse plasma, but diluted, for metabolomics. 15ul plasma in 735ul of water.
Can we work with diluted plasma or can we concentrate it down before processing?
We can concentrate diluted plasma, but 15 µL of plasma is insufficient for conducting metabolite detection. If the sample size is too small, the metabolites within it will be relatively scarce, resulting in many substances being undetectable.
How to work with retinal samples for either Untargeted metabolomics, Widely-Targeted Metabolomics, or lipidomics.
we have experience working with retina samples in TM and untargeted panels. But the report of untargeted panel is unavailable now.
Do you have experiences with fungal supernatant for metabolomics? What are the sample requirements?
We have experience with fungal supernatant for metabolomics.
Sample requirement: Recommended sample size: 100 μl; Minimum sample size: 20 μl; Biological Replicates: ≥ 6
We would like to submit drosophila samples for metabolomics. Would you let me know what recommendation we have for this kind of samples?
We lack prior experience with such complex mixture samples. Therefore, we recommend that clients specify whether they prefer to detect metabolites from the Drosophila body or the liquid component.
We work with plant material and they have performed extraction using ethanol. We intend to send us ethanol extracted solution. Can you work with this extract?
While we can work with this extracts, it's preferable to send us the original sample and allow us to conduct the extraction. This way, we can ensure consistency in sample size by controlling the extraction process.
Are you able to detect microbial metabolites when doing untargeted metabolomics for human plasma? we would like to focus more on microbial metabolomics but at the same time not excluding other metabolites.
We have extensive experience working with human plasma in untargetd metabolomics panel. However, the substances detected represent the total composition of the sample provided, making it difficult for us to differentiate between those originating from human metabolism and those from microbial metabolism.
How to work with dried blood spot before for untargeted metabolomics? We have 300 sample project with dried blood spot.
We have experiences with this sample type. Considering the large sample size involved in this project, we propose conducting a pilot study using experimental and control samples.
We want to analyze purine nucleotides in small pieces of tissue dissected from mouse hippocampus. Is there any metabolomic pipelines we run that is sensitive enough to detect purine nucleotides in limited 1-5mg of brain tissue? The tissue is from control and mutant mice in conventional housing.
The smaller sample size may result in fewer detected substances and decreased data consistency.
How to study metabolomics for a highly threatened manzanita, Arctostaphylos franciscana. We plan to flash-freeze leave tissue in the field with liquid nitrogen and store in the -80ºC freezer until extraction of metabolites.
Just flash freeze in liquid N2 and store in -80C is sufficient. Again, we recommend 600mg of leaf tissue. When they are ready to ship, ship with at least 10 lbs of dry ice.
Do you have any experiences working with hard seeds of wheat? We want to conduct the widely-target panel with the focus on the amino acids within her wheat seeds samples. Do we have any related research with that? I do have the lists of amino acid detected in the leaf samples for wheat from past two projects you provided before.
We have experience working with wheat seeds in Widely-Targeted Metabolomics for Plants. On average, over 200 amino acids have been identified across these three projects. Please see the details in the excel file.
How to submit human T-cells for metabolomics. When collecting the cells, we usually get a number of dead cells among all the cells collected. The question is does the content in the dead cells impact the metabolomics detection results? If so, does cell %viability matter?
Dead cells do not impact the metabolomics detection process. But, in metabolite analysis, we can also detect metabolites from dead cells. Hence, we recommend clients maintain consistency and uniformity in sample collection for precise metabolomics analysis.
We would like to use Widely-Targeted Metabolomics for Plants to assess carbohydrates and use volatile analysis to look into volatiles. 1) Roughly how many carbohydrates are in Widely-Targeted Metabolomics for Plants database? Are you capable of also detecting structural carbohydrates in Widely-Targeted Metabolomics for Plants? 2) For plant volatile analysis, the client wants to know if freezing the flower will change the volatile metabolites in the sample. In other words, comparing volatile metabolomics profile before and after freezing, will there be a major change? If so, what would be best recommended for preserving this kind of samples?
1) The most common structural carbohydrates include polysaccharides and fiber components like cellulose, hemicellulose, and chitin. These macromolecules fall outside the scope of MS detection. 2) There will be minimal alteration in volatile metabolites if samples are stored at -80°C. Sufficient dry ice should be reserved to maintain low-temperature conditions for the samples.
We want to send mouse blood for testing. However, there's quite a lot of hemolysis in the sample and the client wants to do targeted analysis on bile content (secondary bile acid of particular focus). 1) Is it feasible under this condition? By how much will hemolysis affect the effectiveness of this assay? 2) How should the blood samples in this state be prepared and shipped?
Due to hemolysis potentially impacting metabolite levels, we strongly recommend recollecting the samples for optimal metabolomics analysis. However, if re-collection is not possible and you still want to proceed with metabolomics detection on the current samples, we must advise you of the potential risks associated with inaccurate results. Hemolysis can significantly alter metabolite concentrations, leading to unreliable data.
I have an inquiry for bacterial supernatants treated the following ways. Please assess if we can process these sampels: 1). Bacterial cell supernatant, previously filtered by 0.22uM membrane, is precipitated with ethyl acetate (v/v), the pellet is recuperated after drying in ammonium acetate 0.25mM and stored with refrigeration. 2) Bacterial cell supernatant previously filtered by 0.22uM membrane, is precipitated with ammonium sulphate, the pellet is recuperated after drying in ammonium acetate 0.25mM and dialyzed of salts using membrane then stored with refrigeration. 3) Bacterial cell supernatant obtained after grown of bacterial cells in 5% sucrose, exopolysaccharides (EPS) were separated from the cell-free supernatant by the addition of ice-cold absolute ethanol (two volumes) and incubation for 2 days with refrigeration. The precipitated EPSs from liquid culture (EPSQ) were centrifuged and dissolved in double distilled water and dialyzed using a Midi dialysis kit.
The pellet obtained by these methods from the cell supernatant primarily consists of proteins and polysaccharides, which fall outside the scope of MS detection. Therefore, we are unable to process these samples. We can process these liquid samples and attempt to detect metabolites. However, the success of detecting metabolites depends on their presence in the liquid sample. We believe that the recovered pellet mainly comprises proteins and polysaccharides.
We want to submit plant leaves for metabolomics. But they do not have access to liquid nitrogen or lyophilizer. Any advice we can give to the client to best preserve their samples?
So you may collect the sample and keep it in the dry ice and then send the sample to us immediately.
Regarding adherent cell samples: What type of starting material is ideal to accurately capture the metabolite profile of cells, live cells or cell pellets (or either would be okay)?
Frozen cell pellet is best. Minimizes changes in metabolic profile during transit.
For adherent cells, we recommend cells be collected by scraping and not by trypsinizing
Do you have experience on green coffee extract? Which compounds have been detected? Typically green coffee is coffee bean seed that’s not roasted
No, We don't have the experience for the extract, and normally we can handle this kind of sample. How did the client extract the green coffee? from coffee beans or leaf? We have bean and leaf experiences in coffee based on Widely-Targeted Metabolomics for Plants
We want to do widely-targeted metabolomics for some leaf samples from 4-6 different species. About the extraction protocol, would it be okay to send whole leaves (200mg)? And what type of solvent will we use for the extraction?
The minimum sample size for leaf samples in the Widely-Targeted Metabolomics for Plants panel is 300 mg. We advise clients to provide 300 mg for each sample to ensure optimal results. However, if the client is unable to provide larger samples, we can still perform the detection. It's important to note that smaller sample sizes may yield fewer extracted metabolites, affecting the outcome of the analysis. We use 70% methanol for the extraction.
We are interested in the following compounds: UTP, acetylcoA, glutamine, glucosamine.
These 4 compunds all can be detected in TM widely-targeted metabolomics.
How to prepare cell samples? do we need cell pellets or can we also start from lyophilized samples? And what are the shipping conditions?
Cell pellets are recommended and please use dry ice during the shipping process to keep low tempreture for the samples.
We concerns about the impact of washing samples with PBS on their comparability for metabolomics analysis. How does this procedure affect relative quantification.
Please wash the samples with ddH2O after the PBS washing procedure. Then the samples are fine to be used for metabolites detection.
Do you accept Mouse bone marrow for our metabolomic services
yes we do. We have experiences working with bone marrow samples in our TM widely-targeted metabolomics, untargeted metabolomics and quantitative lipidomics panels.
Can you help summerize some project experience of untargeted plus panel on human plasma & serum?
Sure, we have a lot of experience for human plasma & serum metabolomics using widely-targeted metabolomics and untargeted metabolomics.
We are interested in doing SCFA-targeted assay has samples are HIV infected and will need to be handled with BSL2 protocols. Would this project still be feasible considering the Biosafety issues?
For such infectious samples, clients are required to inactivate the samples in advance and must strictly adhere to local regulations for packaging and shipping. It is essential to clearly label potential biohazards on both the outer packaging and sample information form to alert relevant personnel during shipment and experiment. Sales personnel should closely monitor the shipping status of these samples. Upon dispatching the samples, they should promptly provide the receiving center with the contract number, tracking number, sample information form, and details of any potential hazards associated with the samples prior to their arrival.
We are interested in conducting TM Widely-Targeted for Mouse hepatocyte pelleted samples (snap frozen), and we want to know if you normalize the metabolites in each sample by protein concentration or something else?
Yes, we will conduct BCA protein normalization.
Besides the cecal samples I mentioned, we also has some cecal fluid samples which which the extracellular component of mouse cecum suspended into PBS. we have around 200ul. Can you work with this type of sample for targeted BA assay as well?
yes. we can do it. it's fine for the Bile acid targeted analysis.
I am interested in trying your metabolomic analysis and lipidomic profile of murine cells? Do your have experiences in the past with murine cells?
Murine cells are basically mouse cells. We can work with that. The sample requirement is the same as cells.
I have a compound that modulates steatosis that have been testing in a stem cell based model of a high fat diet. I would like to characterize the changes to the metabolomics of the cells upon steatosis induction and to see if the compound is able to return those levels to normal. which assay is best suitable for quantification.
Based on the experiment design provided, we recommend our global metabolite profiling service. Both the widely-targeted metabolomics and untargeted metabolomics assays are suitable options. Untargeted metabolomics assay applies an additional HILLIC column and can better capture compounds with large polarity.
Regarding our Human plasma project, we are worried that the sample collection method may cause interference to the assay. The human plasma samples were obtained the same day from whole blood collected using EDTA tubes, K2 EDTA tubes (BD #366643). Could you please provide some insights about how this preparation process can affect sample metabolites?
The preparation process for plasma samples is correct and appropriate. It will not affect the metabolomics detection. For plasma preparation, firstly collect blood using EDTA tubes and gently invert to mix. Then, centrifuge at 3000 rpm for 10 minutes at 4°C to obtain the supernatant (plasma). Finally, transfer 200μL of the plasma into a new 2mL centrifuge tube for further metabolomics analysis.
Do you have any past project experiences with green algae cultures? These are samples from 10L cultures of Monoraphidium minutum from open-air pond systems. Also what would be the typical sample preparation steps for plants like this.
We have extensive experience working with green algae samples in various panels, including Widely-Targeted Metabolomics for Plants, plant hormone, carotenoids, and anthocyanin. For your reference, we have provided a table showing our experience with green algae samples in the Widely-Targeted Metabolomics for Plants panel. For this type of sample, we recommend a sample size of 40mg, with a minimum of 20mg. If possible, please provide us with the sample collection method so that we can verify its suitability.
I want to conduct a pilot study of examing the function of a protein in macrophages in the lung. I will be submitting mouse cell samples for untargeted plus. Do you have any past experiences relate to this project?
We have several studies using macrophages. We still treat this samples as cells with all the requirements for cells. We have experience working with macrophages using our untargeted metabolomics panel. Macrophages are treated as cell samples, so the sample preparation process is consistent with that of other cell samples.
I am thinking about submitting samples for the analysis of intracellular metabolites. I would like specific sample collection instruction for in vitro cultured primary human hepatocytes.
Sample Collection for Suspended Cells: Collect suitable volume of cell suspension containing 10^6 cells into a 2 mL centrifuge tube. Centrifuge at 1000g at 4°C for 10 minutes, discard the supernatant, and then quickly wash the cell pellet 2 to 3 times with pre-chilled PBS solution (4°C). Centrifuge again at 1000g at 4°C for 10 minutes and discard the supernatant, retaining the cell pellet.
For Adherent Cells: Remove the cell culture medium and wash the cells quickly 2 to 3 times with pre-chilled PBS solution. Add a small amount of PBS solution and gently scrape the cells with a cell scraper, transferring them to a centrifuge tube. Centrifuge at 300g-500g at 4°C for 5 minutes and discard the supernatant. Wash with pre-chilled PBS solution and centrifuge at 1000g at 4°C for 5 minutes, discarding the supernatant. Wash again with PBS solution and count the cell suspension containing PBS. Collect suitable volume of cell suspension containing 10^6 cells into a 2 mL centrifuge tube. Centrifuge at 1000g at 4°C for 10 minutes and discard the supernatant, retaining the cell pellet. Quick-freeze the cell samples in liquid nitrogen for 5-10 minutes, then store them in a -80°C freezer to avoid repeated freezing and thawing.
We have human cancer cell line pellets for untargeted metabolomics. Do you happen to have specific information/cases of the metabolites detected from human cancer cell lines, like the average number, minimum and maximum, metabolites type?
We have extensive experience working with various human cancer cells in our untargeted metabolomics panel. Across these projects, the average, maximum, and minimum numbers of detected metabolites were 2,733, 3,653, and 1,511, respectively. The quantities of each type of detected metabolite are also included in the data. For the untargeted assay, we can provide raw data to the client.
Do you have any experience working with samples of dementia risk before for human patients? It can be serum or plasma.
We have extensive experience with such conventional sample types.
I am interested to study the effect of western diet (high fat/sugar content) in cancer progression using mice models, specifically carcinoma cell line into mouth cavity that later affects oral microbiome. In terms of sample extraction and preparation, what should I do with the swaps? Can you extract from swap or is there a specific protocol I needs to follow? Are you able to examine bacteria metabolites from sliva?
1) For saliva samples collected with swabs, the following requirements apply:
- Sample size: One swab per sample.
- Sample preparation: Saliva should be collected using swabs, immediately placed in liquid nitrogen, and stored at -80℃ for long-term preservation.
After receiving the samples, we will extract the metabolites from the saliva swabs.
2) We can detect metabolites present in both mice and bacteria from saliva samples, but we cannot distinguish whether these metabolites originate from the mouse itself or from bacteria in its mouth. For metabolites specific to bacteria, clients can provide relevant information, and we will assess their detectability using our panels.
I would like to send human Induced pluripotent stem cell (iPSC) for lipidomics. I have an issue with the number of biological replicates required. 30 replicates for each treatment would be too many for in vitro derived models.
We ask for higher replicates for human clinical samples (such as blood, tumor samples, or fecal samples) to allow better statistics accouting for individual variations. For iPSC, we can certainly work with less replicates as cells are grown in a more uniform condition. We would still recommend 6 to have a more robust statistical analyses.
I have research interest on a bacteria actinomycete (Pseudonocardia dioxanivorans), and I am interested in looking at metabolites in the central carbon metabolism cycle. I was wondering if you can provide any recommendations in terms of 1) the types of services that would be best suited, 2) sample types/requirements, 3) sample preparation.
1) Service: the central carbon metabolism is actually the energy metabolism, so we recommend our energy tageted panel and our untargeted plus panel for these metabolites of interest.
2) sample requirements:
recommended sample size: 1*10^10; minimum sample size: 5*10^8.
3) sample preparation: During the logarithmic growth phase of bacteria, bacterial samples are collected. This process begins with measuring the OD600 value of the bacterial culture. Following this, an appropriate volume of culture is transferred to a centrifuge tube based on its concentration and centrifuged at 4000 rpm under 4 degrees Celsius for 10 minutes. After centrifugation, the supernatant is discarded, and the bacterial pellet is washed twice with PBS solution. Finally, the bacterial pellet is stored at a low temperature for metabolomic analysis.
Can you perform Metabolomics on Fungi? If so, how can we deactivate the live Fungi before sending samples in powdered form?
Sure, we can work with fungi samples and have extensive experience.
We have prepared some cell pellets by trypsinization, but there are publications reporting that trypsinization affects the metabolites profile. Do you have any suggestions or standard protocol to collect the pellets from attached cells so that metabolites are well sustained?
If the cells are grown attached to plates, you can collect the cells using cell scrapper.
1. scrap the cells with a little of the media using a cell scraper
2. Pippet the cells into an appropriate tube.
3. Spin down the cells to pellet and aspirate the supernatant.
4. Wash the cells once with PBS
5. Spin down the cells to pellet and aspirate the supernatant.
6. Flash freeze the cells in liquid nitrogen
7. store in frozen cell pellet in -80C until ready to ship.
To obtain an ideal result for analyzing the metabolomics product, what the time period is better after adding drugs to cells? Treatment for 6 h, 12 h or 24 h at 37 ℃?
Well, it depends. Normally, it's 4-8 hours. It would better to analyze the samples after the treatment for 3, 6 and 12 hours.
Do you have past experience on how many metabolites we can identfiy from root exudate? What is the expected number of metabolites for 20mg? What is the expected number of metabolties for 0.5mg?
In our past experiences for untargeted panel, the number of detected metabolites in root exudate has ranged from 1,000+ to 4,000+, depending on the sample.
How to prepare primary cells for metabolomics?
For each sample, take a cell suspension containing at least 10^6 cells and transfer it into a 2 mL centrifuge tube. Centrifuge at 1000g for 10 minutes at 4°C, then discard the supernatant. Quickly wash the cells 2-3 times with pre-cooled PBS solution (4°C), centrifuging at 1000g for 10 minutes at 4°C each time and discarding the supernatant after each wash. Finally, rapidly freeze the cell pellet using liquid nitrogen for 5-10 minutes, then store at -80°C. Ship the samples on dry ice.
We have different patient-derived experimental and control groups. Could you please advise how many samples per group they should submit in this case? 8 or 30?
For human samples, we recommend 30 samples in each group. The more samples there are, the more reliable the experimental results.
How to submit total saliva samples as well as saliva extracellular vesicle samples, does the required sample volume remains the sample across these two samples (500ul recommended, 100ul minimum)?
For saliva samples, the sample size required is 500ul recommended and 100ul minimum. For extracellular vesicle samples, the sample size required is 2*10^9 particles (NTA) recomended and 1*10^9 particles (NTA) minimum.
I want to send EV (extracellular vesicles) to do Untargeted. What buffer should EV be in? Or should the EV be centrifuged as a pellet? Not sure if we can see the EV pellet in this case.
EVs can be preserved in 100 μL of PBS.
What is the feasibility of Targeted Energy Metabolism metablomics on plant samples?
Plant species: Triticum Aestivum; Starting Material: frozen plant tissue
Plant samples are suitable for Targeted Energy Metabolism Metabolomics. The recommended sample size is 300 mg, with a minimum required sample size of 200 mg.
For Plant-Phytohormone Targeted Metabolomics, our samples for this project are lyophilized and ground leaf tissue, what would be the minimum mass required?
Recommended sample size:200 mg;
Minimum sample size: 100 mg
I am interested in submitting endothelial cells for Energy Metabolism and Untargeted Plus Metabolomics. I was wondering if you have any experience performing these panels on supernatant of endothelial cells? If so, do you recommend your costumers to use special culture media or have certain controls, since the media also contains metabolites?
We have extensive experiences working with cell supernatant samples in both Untargeted and Energy-Metabolism Targeted panels. There are no any special requirements for the culture media. If the goal of metabolomics detection is to compare differential metabolites between the experimental and control groups, providing blank controls is unnecessary since both groups use the same culture media. However, if background metabolites in the culture media need to be eliminated during data analysis, at least three blank media controls should be provided.
We want to test the following analytes: trans-B-carotene, 13-cis-B-carotene, a-carotene, B-cryptoxanthine, Lutein, Zeaxanthine, Lycopene, Retinol, a-tocopherol, y-tocopherol. These would be in an EDTA sample matrix for human samples.
For these ten compounds, our targeted carotenoid panel can cover six of them. Although the untargeted panel can also cover most of these compounds, the identification levels are 2 or 3, meaning the identifications are not very accurate. We recommend using the carotenoids targeted metabolomics panel.
Our samples are organoids, and it's challenging to calculate cell numbers accurately. I was wondering what would be the alternative methods for normalization besides cell counting?
Protein quantification can be used as an alternative method of normalization for organoid samples. At least 10 µg of protein is required for each sample.
Do your accept mouse feces samples as starting materials? We focuse on energy metabolism and short chain fatty acids. What would be the sample requirement?
Yeah, we accept mouse feces to conduct energy targeted and SCFA targeted metabolomics. We recommend 200 mg samples for the analysis, at least 20 mg.
Hey guys, For the energy metabolism panel, could you perform enrichment analysis on Carbon 13 fractions for downstream metabolites of glucose and pyruvate (i.e., lactate, glutamate, etc.)? Additionally, we are interested in the quantification of high energy phosphates, including: NAD, NADH, NAD/NADH, AMP, ADP, ATP, Total AMP/ATP, ADP/ATP, EC, Acetyl CoA, Mal CoA, Suc CoA, Free CoA, Total CoA, Free CoA/Bound CoA.
Our final report includes functional enrichment analysis. For compounds present in the database, we can perform enrichment analysis for the client. Our energy-metabolism targeted metabolomics can detect NAD, AMP, ADP, ATP, Acetyl CoA, and Suc CoA.
For mouse stool pellets, is there any specific method for sample collection and preservation, or is snap freeze sufficient enough?
Mouse/Rat Fecal Collection Method: Place the mouse or rat in a clean cage lined with sterilized filter paper. After defecation, promptly collect the feces using sterile tweezers, mix the sample thoroughly, and transfer it to a sterile cryovial (at least 200 mg per vial). Quickly freeze the sample in liquid nitrogen for 5-10 minutes, then store it in a -80°C freezer. Avoid repeated freeze-thaw cycles.
Recommended Sample Quantity: Generally, 6-8 pellets of mouse feces or 1-2 pellets of rat feces are required.
For metabolomics sample preparation, should I collect the stool and homogenize them in a liquid? Water?
Simply collect the stool samples in tubes and store them at -80°C in fine.
We would like to study the metabolomics of cancer in mouse. They would like to know the fatty acids, amino acids, and carbohydrate metabolites, but we are not sure of the specific metabolites that may focus on. Which pipeline is more appropriate, untargeted or widely-targeted metabolomics?
If the metabolites interested in include many highly polar metabolites, such as amino acids and carbohydrates, an untargeted metabolomics approach using a HILIC column would be fine.
We are interested in your Energy Metabolism and Widely Targeted Metabolomics platform. We have mouse tumor adherent cells that are hard to scrape. Without using trypsin, are there any other recommendations we can provide for us to get the cells off the plate?
Here is an alternative sampling method for adherent cells:
1) Quickly discard the culture medium and invert the dish onto absorbent paper to remove any remaining liquid.
2) Add pre-chilled PBS (4°C) and rinse the cells 2-3 times. If using a pipette, add the PBS along the wall of the dish to avoid dislodging the cells. Discard the PBS.
3) Use a pipette to remove any residual PBS, then quench the cells by touching the bottom (outer wall) of the dish with liquid nitrogen.
4) Add 500 µL of pre-chilled methanol-water (4:1, v/v) to the dish, scrape the cells with a cell scraper, and transfer the suspension to a 1.5 mL centrifuge tube using a pipette.
5) Add another 500 µL of pre-chilled methanol-water (4:1, v/v) to the dish and transfer any remaining cells to the same centrifuge tube.
6) Seal the tube with parafilm and store it at -80°C.
Note: After using this method for cell sampling, the samples cannot be used for protein quantification or cell counting. you should perform cell counting in advance and ensure consistent sampling. The recommended sample size is 1 × 10^7 cells, with a minimum of 1 × 10^6 cells.
We are interested in either our Untargeted metabolomics or TM Widely Targeted panels. We are looking to identify molecules related to glycosylation, like carbohydrates, as well as reproductive hormones (estrogen, testosterone, estradiol). We are concerned about sample volume. Would 20-30 µL of mouse serum be enough for the analysis to effectively detect both hormones and carbs
Both the widely-targeted metabolomics and untargeted metabolomics panels are suitable for detecting these two types of metabolites. A sample of 20-30 µL of serum is sufficient for either TM or Untargeted metabolomics analyses. We extract metabolites from serum using 70% methanol.
Are there any suggestion for collecting plasma samples? The blood samples are taken from heart, and sometimes will cause hemolysis. What method is best for collecting plasma for metabolomics analysis?
Our sample submission guide provides detailed instructions on plasma sampling. Ensuring that plasma samples are free of hemolysis before submission is crucial, as hemolysis can significantly affect the metabolic profile. We recommend isolating plasma promptly after whole blood collection, ideally within 30 minutes, and aliquoting it into multiple 2 ml centrifuge tubes for storage at low temperatures. This method maximizes sample usability and minimizes the risk of degradation due to repeated freeze-thaw cycles.
Are you able to work with Skin Swabs for our untargeted metabolomics panel?
We accept swab samples with one swab per sample, ensuring consistency in the sampling process. After collection, place the swab in an appropriate tube, snap-freeze it in liquid nitrogen for 5-10 minutes, and store at –80°C.