Lipidomics Sample Preparation FAQ

How to prepare lipid droplets/ liposomes?

Preparing lipid droplets or liposomes begins with choosing the right lipid sources. For lipid droplets, cells can be cultured under conditions that promote lipid accumulation, such as in the presence of fatty acids. Once the cells have grown, harvest them by centrifugation and wash with PBS to remove excess media. For liposome preparation, mix lipids (like phospholipids) in a suitable organic solvent, then evaporate the solvent under reduced pressure to form a lipid film. Hydrate this film with an aqueous buffer to form liposomes, which can then be sized by extrusion through filters. If isolating lipid droplets from cells, you may need to lyse the cells with a lysis buffer, then centrifuge to separate the lipid droplets based on their buoyancy. Store isolated lipid droplets or liposomes at -80°C for further analysis.

How to prepare sebum on the skin collected by Sebutape patches?

To prepare sebum samples collected using Sebutape patches, first ensure the skin is clean and dry before application. Apply the Sebutape patch to the target area, pressing firmly to ensure good adhesion. Leave it on for the recommended time to allow for adequate sebum absorption, typically around 30 minutes to an hour. After removal, carefully place the Sebutape patch into a sterile container to prevent contamination. If immediate analysis is not possible, freeze the patches at -80°C to preserve the sebum components. When ready for analysis, you can extract the sebum from the tape using organic solvents, such as chloroform or methanol, to isolate the lipids for further metabolomic profiling.

What is the best temperature for long-term storage of metabolomics/lipidomics samples?

The best temperature for long-term storage of metabolomics and lipidomics samples typically ranges from -80°C to -20°C, depending on the stability of the specific metabolites or lipids of interest. Storing samples at -80°C is often ideal, as this ultra-low temperature helps preserve the integrity of sensitive compounds by minimizing enzymatic activity and degradation. For example, fatty acids and certain polar metabolites are particularly susceptible to oxidation and hydrolysis, so maintaining low temperatures is crucial to prevent these processes.

How can I minimize enzymatic activity during sample collection to prevent degradation of metabolites/lipids?

One effective approach is to use appropriate anticoagulants, such as EDTA or heparin, which can inhibit enzymatic activity in blood samples. Additionally, immediately placing samples on ice or using cryopreservation techniques can help slow down metabolic processes and preserve sample integrity. Another strategy is to rapidly process samples after collection. For example, if working with tissues, quickly snap-freezing the samples in liquid nitrogen can halt enzymatic reactions. For liquid samples like plasma or serum, processing the samples to separate the liquid phase from cells as quickly as possible can also prevent metabolite degradation caused by cellular enzymes.

What is the effect of exercise on metabolite or lipid profiles in collected samples?

Exercise has a significant impact on metabolite and lipid profiles, leading to dynamic changes in the concentrations of various compounds. For instance, during physical activity, the body undergoes metabolic shifts to provide energy, resulting in increased levels of fatty acids, lactate, and certain amino acids. Research has shown that prolonged exercise can enhance the mobilization of lipids from adipose tissue, leading to higher circulating levels of free fatty acids, which are used as fuel by muscles. Moreover, exercise can also affect the metabolism of carbohydrates, with increased glucose uptake by muscle cells. This shift can lead to changes in metabolite levels, such as elevated lactate during high-intensity exercise and altered levels of various metabolites associated with the Krebs cycle. These metabolic changes are important for understanding how exercise influences overall health and can inform strategies for optimizing performance or recovery in athletic populations.

How does sample pH influence metabolite or lipid stability during collection?

Sample pH plays a critical role in the stability of metabolites and lipids during collection. Many metabolites are sensitive to pH changes, which can affect their ionization state and overall stability. For example, amino acids can undergo degradation or oxidation if the pH is not optimal, leading to altered concentrations. Maintaining a neutral pH (around 7.0) is often recommended for preserving the integrity of most metabolites.

How should lipid extraction be performed to prevent oxidation?

To prevent oxidation during lipid extraction, it’s essential to use an extraction method that minimizes exposure to oxygen. One effective approach is to perform the extraction in an inert atmosphere, such as nitrogen or argon, which displaces oxygen. For example, we might carry out the extraction in a glove box filled with inert gas or use sealed containers during the process. This helps ensure that sensitive lipids, such as polyunsaturated fatty acids, remain stable and intact. Additionally, incorporating antioxidants into the extraction solvent can further protect lipids from oxidative degradation. Common antioxidants include butylated hydroxytoluene (BHT) or ascorbic acid, which can scavenge free radicals and prevent lipid peroxidation. Adding BHT to a chloroform-methanol extraction solvent can help maintain the integrity of extracted lipids.

What is the role of phase separation in lipid extraction protocols?

Phase separation is a crucial step in lipid extraction protocols, as it helps isolate lipids from other cellular components in a sample. Typically, a solvent mixture, such as chloroform and methanol, is used to extract lipids from biological matrices. When the mixture is added to the sample and shaken, it creates distinct phases: a lower organic phase containing the lipids and an upper aqueous phase that retains polar metabolites and other biomolecules. This separation allows for selective recovery of lipids without interference from other compounds. When extracting from tissues, we often need to optimize the volume ratios of solvents to maximize lipid recovery. Proper phase separation also minimizes the risk of co-extracting unwanted substances, such as proteins or sugars, which can complicate subsequent analyses.

How to prevent lipid degradation during extraction?

Preventing lipid degradation during extraction involves implementing strategies that minimize exposure to heat, light, and oxygen. One effective method is to perform the extraction at low temperatures, such as on ice or at 4°C, to reduce enzymatic activity and chemical reactions that could lead to degradation. Using antioxidants in the extraction solvent is another critical strategy. Incorporating substances like ascorbic acid or butylated hydroxytoluene (BHT) can protect lipids from oxidation during extraction. Additionally, rapid processing is vital; samples should be extracted as soon as possible after collection to prevent any metabolic changes.

Do we have experience with bacterial extra cellular vesicles? But we would still be able to run the quantitative lipidomics assay on this sample type, correct?

We can run the quantitative lipidomics assay on this sample type, and we need 2x10^9 particles, BCA protein total 40ug. Minimum sample size (not accepted below this sample size): 1×10^9 particles (NTA), total BCA protein 20ug.

What would be our sample requirement for tissue homogenate samples in quantitative lipidomics?

Attention Points: (1) When dealing with large tissues such as the liver and pancreas, it is recommended to collect samples from multiple points and combine them in a single sterile centrifuge tube to guarantee consistent sampling across various tissue regions. (2) Maintain uniformity in the sampling location for systemic tissues like muscle. (3) For esophageal and adipose tissues, collect samples following an overnight fast. (4) Conduct tissue sample processing and cutting swiftly on ice to prevent degradation, as prolonged exposure could compromise sample integrity. Recommended Guidelines for Samples: Sample Size: 300 mg; Biological Replicates: Human samples ≥ 30/group; Animal samples ≥ 8-10/group;

We are interested in our Quantitative Lipidomics analysis for neural tissue, and has samples from 10-25mg of weight. Can we run samples with that low of volume?

We require a minimum sample size of 20 mg for normal tissues in lipidomic analysis . It is necessary for the client to ensure that each sample meets this requirement before we can proceed with the testing.

We want to send bacterial cells and bacterial EVs (extracellular vesicles) for Quantitative Lipidomics. Do these two cell types uses the same extraction method? Can the data from these two sample type be combined and compared?

These two cell types use the same extraction method and the data can be combined and compared.

We would like to detect lipidome profile on renal organoid. Do we have experiences on renal organoid? They focus on prostaglandin metabolism, such as PGE2, 15-keto-PGE2 and 13,14-dihydro-15-keto-PGE2.

While we do not have direct experience with renal organoids, we do have expertise in working with renal tissue in our QL panel. Within this panel, we can detect several types of prostaglandins and numerous other lipids. For detailed information, please consult the details as provided in the Excel file.

I have a client that will extract nuclei from Arabidopsis to do Quantitative Lipidomics for Plants and study nuclear envelope. What is our sample requirement if the client sends nuclei?

recommended sample size: 1*10^7 nuclei minimum sample size: 1*10^6 nuclei

For lipidomics analysis, do we still require 3 technical replicates per clone for cell line samples? How many sets of samples need to be tested in total?

We recommend that clients provide at least three duplicates (either biological or technical) for each sample in any panel. This ensures that we can thoroughly analyze the data and provide a comprehensive report after the detection process is completed. In the case you described, it appears that there are four biological duplicates for each treatment in each cell line. Therefore, the client can send at least three wild-type (WT) and three knockout (KO) samples for each cell line, totaling (3 + 3) * 2 = 12 samples for their testing requirements.

I want to do lipidomics on isolated organelles membrane from human cells. What buffer would we recommend the client to preserve their samples? we usually use 10% or 5% FBS in PBS. Would that be acceptable with your process?

FBS is not suitable for preserving samples for metabolomics detection because it contains metabolites. PBS is acceptable, but it is preferable to pellet the samples and send the pellets to us for metabolomics detection.

I want to do Quantitative Lipidomics on Olive oil. Can we process these samples? If yes, how much oil do we need?

We can process this type of sample, 1ml is fine.

Can you show some demo data on Quantitative Lipidomics where plasmalogens have bene well captured? Also do you have a demo data on Tryptophan Targeted panel where tryptamine is well captured?

The plasmalogens we can detect in our QL panel are PE-Ps. Please refer to the demo data from a project working with mouse cells, where over 100 PE-Ps were detected. Additionally, demo data from a project working with mouse feces in the tryptophan-targeted panel could be provided.

I am interested in submitting spectral supernatants samples to our quantitative lipidomics panel. how to prepare the samples?

Supernatant Sample Collection Method: Collect cell supernatant in a sterile centrifuge tube. Centrifuge at 1200 rpm for 5 minutes at 4°C to remove cell debris and impurities. Using a sterile syringe, draw the supernatant and filter it through a disposable 0.22 μm syringe filter into a new centrifuge tube to remove any remaining bacteria. 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 Supernatant Sample Volume: 100 µl.