Preparation of Human Tissues Embedded in Optimal Cutting Temperature Compound for Mass Spectrometry Analysis

Podsumowanie

Sphingolipids are bioactive metabolites with well-established roles in human disease. Characterizing alterations in tissues with mass-spectrometry can reveal roles in disease etiology or identify therapeutic targets. However, the OCT-compound used for cryopreservation in biorepositories interferes with mass-spectrometry. We outline methods to analyze sphingolipids in human tissues embedded in OCT with LC-ESI-MS/MS.

Streszczenie

Sphingolipids are cellular components that have well-established roles in human metabolism and disease. Mass spectrometry can be used to determine whether sphingolipids are altered in a disease and investigate whether sphingolipids can be targeted clinically. However, properly powered prospective studies that acquire tissues directly from the surgical suite can be time consuming, and technically, logistically, and administratively challenging. In contrast, retrospective studies can take advantage of cryopreserved human specimens already available, usually in large numbers, at tissue biorepositories. Other advantages of procuring tissues from biorepositories include access to information associated with the tissue specimens including histology, pathology, and in some instances clinicopathological variables, all of which can be used to examine correlations with lipidomics data. However, technical limitations related to the incompatibility of optimal cutting temperature compound (OCT) used in the cryopreservation and mass spectrometry is a technical barrier for the analysis of lipids. However, we have previously shown that OCT can be easily removed from human biorepository specimens through cycles of washes and centrifugation without altering their sphingolipid content. We have also previously established that sphingolipids in human tissues cryopreserved in OCT are stable for up to 16 years. In this report, we outline the steps and workflow to analyze sphingolipids in human tissue specimens that are embedded in OCT, including washing tissues, weighing tissues for data normalization, the extraction of lipids, preparation of samples for analysis by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), mass spectrometry data integration, data normalization, and data analysis.

Wprowadzenie

Sphingolipids are bioactive metabolites known for their roles in human metabolism and disease^1,2. They regulate complex cellular processes such as cell migration, cell survival and death, cell movement, vesicular trafficking, cellular invasion and metastasis, angiogenesis, and the production of cytokines^{1,2,3,4,5,6,7,8,9}. Defects in the regulation of sphingolipid metabolism contribute to the initiation and progression of cancers, determine how aggressive cancers are, and how cancers respond to and develop resistance to therapy^3,10. Therefore, because of these broad impacts on the etiology of disease, analytical methods that can precisely establish disease-specific sphingolipid alterations are important tools. Mass spectrometry (MS) is the most accurate and reliable method to analyze sphingolipid alterations.

Human specimens that can be used for the analysis of sphingolipid alterations can be obtained prospectively from the surgical suite or retrospectively from tissue biorepositories. Fresh tissues from surgery are advantageous because they can be directly analyzed by MS or other analytical methods. However, acquiring tissues prospectively has administrative, technical, and logistical hurdles, and collecting sufficient specimens to reach statistical power can be challenging. Obtaining tissues from biorepositories is advantageous because they can be acquired retrospectively, in large numbers, and biorepositories confirm histology and pathology, use standard operating procedures to cryogenically preserve and store tissues, and can provide clinicopathological data that can be used for correlation analyses. However, to preserve molecular and structural features, biorepositories may cryopreserve tissues by embedding them in optimal cutting temperature (OCT) compound, which we have shown interferes with data normalization assays and the quantification of sphingolipids by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS)¹¹. It has also been shown that polyvinyl alcohol and polyethylene glycol, the primary components in OCT compound, result in ion suppression in other MS analysis platforms^12,13,14,15. Therefore, OCT compound must be removed from tissues prior to sphingolipidomic analysis by MS.

In a previous report, we have validated a protocol for the removal of OCT compound from human specimens for LC-ESI-MS/MS analysis¹¹ and the methodology used for weighing tissues for data normalization¹¹. Here, we detail the steps of the sphingolipidomic OCT compound-removal protocol (sOCTrP) and show representative data from human lung adenocarcinoma tumors and normal adjacent uninvolved tissues.

Protokół

De-identified human lung tissues were obtained from the Virginia Commonwealth University (VCU) Tissue and Data Acquisition and Analysis Core under an internal review board (IRB) approved protocol (#HM2471). The use of mice for research and harvesting of mice tissues was approved by the VCU institutional animal care and use committee (IACUC).

1. Preparation of materials

NOTE: These steps should be performed a day prior to the tissue washing.

1. Pre-label and weigh 1.5 mL polypropylene centrifuge tubes with concise but clear identifier codes for each sample that will be processed the next day. Use a chemically resistant permanent marker (see Table of Materials) that will not fade under the repeated tube handling.
2. Open an electronic spreadsheet and label consecutive columns with the following column headers: tube identifier (ID), tube alone, tube w/tissue, and total tissue. Enter the tube identifiers that will be processed into the column labeled tube ID.
3. Pre-calibrate and zero an analytical scale. Place a clean 10 mL Erlenmeyer flask in the center of the balance plate (the flask will act as a tube holder). Close the weighing chamber door and tare the scale with the flask.
4. Weigh 1.5 mL centrifuge tubes. Carefully place the tube in the flask and close the weighing chamber door. Allow the weight to stabilize and record the weight in the column labeled tube alone.
   NOTE: Avoid moving the 10 mL Erlenmeyer flask when weighing tubes. If the flask is moved, re-center the flask and re-tare the balance. Place the closed tubes in a rack and store until needed.
5. Pre-label 13 x 100 mm screw-top glass tubes and caps with the identifier codes and fill them with 2 mL of LC-MS grade methanol. Place them in a tube rack, cap the tubes, and store them in a refrigerator (4 °C) until use.
   NOTE: Use a bottle top solvent dispenser (see Table of Materials). If one is not available, use glass pipettes to avoid the use of plastics when dispensing organic solvents.
6. Pre-label 13 x 100 mm glass test-tubes. Cover and store the test tubes at room temperature.
7. Pre-label autoinjector vials with identifier codes. Cover and store the autoinjector vials at room temperature until use.
8. Prepare tissue-drying wicks.
   1. Begin by cleaning surgical scissors by immersing them in LC-MS grade methanol for 5 min, and then wipe them with a clean laboratory grade tissue.
   2. Using powder free gloves, twirl the end of a laboratory grade tissue (see Table of Materials) until a finely tapered 30-40 mm wick is formed.
   3. Using methanol-cleaned scissors, cut the wick on the thick end. Place wicks in a clean carboard box. Make double the number of wicks than will be needed to process all the samples.
9. Prepare shortened 1 mL pipette tips. Using methanol-cleaned surgical scissors, cut 4-5 mm off the tip of a 1 mL pipette tip, and place the tip back in the tip holder. Prepare twice as many tips as samples to be processed.
   ​NOTE: These will be used in the retrieval of washed tissues from tubes. Do not touch the end of the tip.
10. Pre-chill molecular biology grade phosphate buffered saline (PBS) to 4 °C (0.5 L is sufficient). This will be used to retrieve specimens. For each sample that will be processed, pre-chill (4 °C) 60 mL of ultra-pure deionized water.
11. Prepare mass spectrometry internal standards. Dissolve the following lipids in ethanol:methanol:water (7:2:1; v/v/v) at a concentration of 25 nmol/mL: d17:1-sphingosine, (2S,3R,4E)-2-aminoheptadec-4-ene-1,3-diol; d17:1-sphingosine-1-phosphate, heptadecasphing-4-enine-1-phosphate; C12-Ceramide (d18:1/C12:0), N-(dodecanoyl)-sphing-4-enine; C12-sphingomyelin (d18:1/C12:0), N-(dodecanoyl)-sphing-4-enine-1-phosphocholine; C12-glucosylceramide (d18:1/C12:0), N-(dodecanoyl)-1-β-glucosyl-sphing-4-eine; C12-lactosylceramide (d18:1/C12:0), N-(dodecanoyl)-1-ß-lactosyl-sphing-4-ene.

2. Tissue washing

NOTE: Completing all steps in section 1 one day prior allows a skilled researcher to process up to 40 samples per day, and novices ~10-15 samples per day. Begin early in the morning and do not stop until all steps in sections 2.1-3.8 are completed.

1. On the day that the tissues will be processed, prepare the biosafety cabinet work area by equipping it with a vortexer, a fully charged serological pipettor, and an ice tray. For all steps in this section, wear appropriate PPE, including a lab coat, a double layer of gloves, and protective eyewear. CAUTION: Human tissues and fluids should be considered and treated as biohazardous materials.
2. Pre-chill (4 °C) a swinging bucket centrifuge, any adapters needed for holding 15 mL conical centrifuge tubes, and centrifuge biocontainment lids. Pre-chill (4 °C) a fixed angle centrifuge capable of holding 1.5 mL centrifuge tubes.
3. If tissues are not aliquoted into 15 mL polypropylene centrifuge tubes, use a methanol cleaned spatula (clean for each new tissue sample) to transfer them to pre-labeled 15 mL polypropylene tubes. Label the 15 mL caps also.
   1. Place a tube rack capable of holding 15 mL centrifuge tubes in an ice tray and fill the tray with ice. Thaw tissues that will be processed that day by placing them in the rack on ice.
      NOTE: A minimum of 3-4 mg of tissue should be requested from the biorepository for processing as it has been previously shown that tissue washing and weighing protocols are accurate and reliable at these weights¹¹.
4. Move the ice tray with the samples into the biosafety cabinet.
5. Perform three sOCTrP cycles¹¹ as discussed below (i.e., perform steps 2.5.1-2.5.5 three times).
   1. Add 10 mL of ice-cold ultra-pure deionized water to each tube and cap them tightly. Allow the tubes to incubate for 10 min.
   2. Vigorously vortex each tube for 10-20 s or until all the tissue deposited on the walls of the tube, or a tissue pellet, is completely resuspended.
      NOTE: In sOCTrP cycles 2-3, it is critical that the pellet be resuspended so that any OCT in the pellet is diluted into the wash solution.
   3. Transfer the tubes to the pre-chilled (4 °C) swinging bucket centrifuge. Centrifuge samples at 4,000-5,000 x g for 10 min at 4 °C.
      NOTE: Avoid the generation of and exposure to biohazardous aerosols generated during centrifugation by sealing the centrifuge carriers with a biocontainment lid (see Table of Materials).
   4. Retrieve samples from the centrifuge and transfer to the ice tray. Place the tray in a biosafety cabinet and uncap the tubes. Save the caps.
   5. Carefully aspirate the wash solution. Avoid aspirating the tissue material in the pellet by leaving ~0.5 mL of the supernatant in the tube. Cap the tubes and avoid cross-contamination by matching labeled caps with tubes.
      ​NOTE: The wash solution (the supernatant) in step 2.5.5 is a biohazardous material; handle and dispose of it following biosafety guidelines appropriate for specimens of human origin.

3. Tissue weighing (for data normalization)

1. After the last sOCTrP wash cycle, carefully aspirate most of the wash solution but avoid disturbing the pellet. Leave ~0.5 mL of the wash solution in the tube.
2. Using the shortened pipette tips (prepared in step 1.9), take-up 500 µL of ice-cold PBS and add it to the tube.
   1. Using the same tip, gently resuspend the pellet and retrieve all tissue. Avoid turbulent pipetting to decrease tissue loss by its adherence to the tube and pipette tip walls.
3. Add the resuspended tissue to its corresponding pre-labeled and pre-weighed 1.5 mL centrifuge tube and place the tube on ice. Repeat for all tissues in the data set.
4. Place the 1.5 mL tubes into a pre-chilled centrifuge and pellet the tissues at 7,000 x g for 5-7 min and 4 °C.
   1. Retrieve tubes and carefully aspirate as much of the PBS as possible without disturbing the pellet. Place the tube back on ice.
5. Centrifuge tubes for an additional 3 min at 7,000 x g to ensure tissues are well pelleted. Transfer the tubes to ice.
   NOTE: The additional centrifugation step is important to prevent tissue loss when removing all wash solution and wicking. If processing more than five samples, repeat the 3 min, 7,000 x g centrifugation step after every fifth sample is processed to ensure tissues remain pelleted.
6. Using the wicks prepared in step 1.8, gently remove any remaining wash solution. Use a clean wick for each sample. It is acceptable to gently dab the tissue with the wick, which will help remove most of the wash solution but avoid losing any tissue by adherence to the wick. Close the tube and place it on ice.
7. Weigh each tube in the recently calibrated, tared, and zeroed analytical balance.
   1. Place each tube in the glass Erlenmeyer flask and close the chamber door. When the weight has stabilized, record it in the electronic spreadsheet column labeled tube w/tissue.
   2. After weighing, place the tube back on ice.
8. Add 300 µL of ice-cold PBS. Using a shortened pipette tip (step 1.9), carefully retrieve all tissue and deposit in a pre-labeled screw-top glass tube (prepared in step 1.5) containing 2 mL of ice-cold LC-MS grade methanol (add the tissue to the methanol, not to the side of the tube).
9. Calculate the amount of tissue that was washed by subtracting the tube alone from the tube w/tissue value. Record this number in the total tissue column. The total tissue value will be used to normalize mass spectrometry data in step 6.12.
10. Store the samples at -80 °C until ready to perform steps 4.1-4.15.
    ​NOTE: This is a good stopping point, and the samples can be stored safely for a couple of weeks at -80 °C. For other tissue normalization methods, such as protein concentration or lipid phosphate content, refer to Rohrbach et al.¹¹.

4. Lipid extraction

NOTE: It is important to begin these steps in the morning, particularly when many samples will be processed. Before starting, pre-heat a water bath or incubator to 48 °C.

1. Retrieve the samples prepared in step 3.8 and the MS lipid standards (prepared in step 1.11) from the freezer. Allow them to equilibrate to room temperature for 20 min.
2. Vortex and immerse the internal standard solution in an ultrasonic water bath (see Table of Materials) for 2-3 min or until the MS lipid standards are fully dissolved prior to dispensing into samples. This will avoid errors in data normalization.
3. Using a repeating pipette, add 250 pmol (10 µL from the 25 nmol/mL solution prepared in step 1.11) of internal mass spectrometry standards to each tube. Re-cap the tubes lightly.
4. To facilitate homogenization, adjust the methanol volume to 2 mL. Only use LC-MS/MS grade methanol.
5. Working one tube at a time, use a homogenizer to triturate the tissue until no clumps are visible (typically 5-20 s). Move the homogenizer tip in a circular motion and gently press against the tube wall to assist trituration. Re-cap the tube.
   NOTE: Ensuring all tissues are finely triturated will maximize lipid extraction. Do not add chloroform to samples prior to homogenization as the disposable homogenizer tips are made of plastic that will dissolve in solutions containing chloroform.
6. Immerse the tube at a 45° angle in an ultrasonic water bath for 5-10 s.
   1. If no tissue clumps form upon immersing in the ultrasonic bath, cap it, and move on to the next tube.
   2. If tissue clumps form when the tube is immersed in the ultrasonic bath, re-homogenize, and target the clumps.
   3. Repeat this process (homogenization/immersion in an ultrasonic bath) iteratively until all large tissue clumps are broken-up.
7. In a fume-hood, add LC-MS grade chloroform and methanol to each tube (use a bottle top dispenser) to achieve a ratio of 2:1:0.1 methanol:chloroform:water. Using clean caps, seal the tubes tightly and leave on the benchtop until the end of the day.
   1. For tissues weighing 50 mg or less, use a total extraction volume of 4 mL, and adjust using this ratio (50 mg:4 mL extraction solution) for larger specimens.
8. Before leaving that evening, place the tightly capped tubes in a 48 °C water bath or incubator and incubate them overnight.
   NOTE: It is critical that the tubes be tightly capped so that the solvent ratios do not change due to evaporation.
9. The next morning, retrieve the samples from the 48 °C water bath and pellet any solvent-insoluble debris by centrifugation at 4,000-5,000 x g at 4 °C for 20 min.
10. Working in a fume-hood, carefully decant the supernatant into the pre-labeled 13 x 100 mm borosilicate glass test-tubes. Tilt the 13 x 100 mm tube at a 30-45° angle to facilitate decanting.
    NOTE: If processing more than 12 samples, do not allow the centrifuged tubes to sit for extended periods of time as the pellets will soften and insoluble debris will be transferred when performing step 4.10. To avoid this, only take 12 tubes out of the centrifuge at a time and continue centrifuging the other tubes until you are ready to decant them.
11. Transfer the tubes to a vacuum concentrator capable of holding 13 x 100 mm tubes. Evaporate all solvent with an initial 1 h heating step (40 °C) and run under the maximum vacuum.
12. Remove tubes from the vacuum concentrator and add 500 µL of LC-MS grade methanol (use a bottle top dispenser) to each tube.
13. Resuspend the dried lipid extracts by vortexing for 5-10 s, followed by immersing tubes at a 45° angle in an ultrasonic water bath for 2 min while rotating the tube. Re-vortex for 5 s and immerse in ultrasonic water bath for an additional minute.
    NOTE: This is a critical step to ensure the maximal recovery of extracted lipids. Do not move ahead until all the dried material on the wall of the tube has been resuspended.
14. Place the 13 x 100 mm tubes in a pre-chilled (4 °C) centrifuge and remove any insoluble debris by centrifugation at 4,000-5,000 x g at 4 °C for 20-30 min.
15. Carefully decant the clarified supernatant into a pre-labeled autoinjector vial. Tilting the autoinjector vial at a 30-45° angle will facilitate decanting. Ensure that the entire contents of the 13 x 100 mm tube are decanted into the autoloader vial.
16. Cap the tubes tightly and store tubes at -80 °C until analysis by LC-ESI-MS/MS.

5. LC-ESI-MS/MS analysis

NOTE: The following procedure uses a binary pump system coupled to an autoinjector, degasser, and LC-MS/MS system operating in a triple-quadrupole mode. The column temperature was maintained using a column oven. See Table of Materials for details on the equipment used.

1. Prepare mobile phase A1 (CH₃OH:H₂O:HCOOH, 58:41:1, v:v:v, with 5 mM ammonium formate) and mobile phase B1 (CH₃OH:HCOOH, 99:1, v:v, with 5 mM ammonium formate). Use only LC-MS grade solvents, water, and reagents.
2. For each set of samples, prepare four Blank autoloader vials containing 500 µL of LC-MS grade methanol.
3. For each set of samples prepare two Internal Standard (label as IS) autoloader vials by diluting 10 µL (250 pmol total each standard) of the internal standard solution prepared in step 1.11 in 500 µL of LC-MS grade methanol.
4. Set the column oven temperature to 60 °C, and the ion source temperature to 500 °C.
5. For the MS/MS analysis, utilize the triple-quadrupole mode of the spectrometer. Set the first quadrupole (Q1) to pass precursor ions, while setting the third quadrupole (Q3) to pass molecularly distinctive product ions (or a scan across multiple m/z in Q1 or Q3) using N2 to collisionally induce dissociations in quadrupole 2 (Q2), which is offset from Q1 by 30-120 eV.
6. Set the multiple reaction monitoring pairs (MRM) detection window at 120 s with a target scan time of 1.0 s. Refer to Table 1 for a listing of MRM pairs, ionization energies, and collision energies.
7. Use the following parameters to resolve sphingolipids in the LC step: using a flow rate of 0.7 mL/min, equilibrate a 2.1 (i.d) x 50 mm C18 reverse phase column for 0.5 min with a solvent mixture of 95% mobile phase A1 and 5% mobile phase B1.
   1. After sample injection, maintain the mobile phase A1/B1 ratio at 95/5 for 2.25 min, followed by a linear gradient to 100% B1 over 1.5 min, which is then held at 100% B1 for 5.5 min, followed by a 0.5 min gradient return to 95/5 A1/B1.
   2. After the run, re-equilibrate the column with a mixture of 95/5 A1/B1 for 0.5 min.
8. Use the following settings for the sample autoloader: rinsing volume, 500 µL; needle stroke, 52 mm (this should be adjusted depending on vial size and volume in each vial); rinsing speed, 35 µL/s; sampling speed, 5.0 µL/s; rinse dip time, 2 s; rinse mode, before and after aspiration; rinse time,2 s.
9. Place samples prepared in step 4.15 onto an autoloader tray in the following order: Blank; IS; Blank; Samples prepared in step 4.15; Blank, IS, Blank.
10. Analyze 5-10 µL of each sample by LC-ESI-MS/MS. Analyze the same volume for all samples in the data set.

6. Data processing, integration, and normalization

NOTE: Although the following steps outline a procedure for specific software (see Table of Materials), similar procedures on equivalent LC-MS instruments and software can be used to integrate MRM pairs for the analyzed lipid classes and corresponding internal standards.

1. Open the quantitation software. In the Quantitate tab double click on the Quantitation Wizard. In the pop-up window, in the left-most workspace, navigate to the correct data set and left click on it. The available samples will be shown in the middle workspace.
2. Highlight the samples to be analyzed and then click on the right-handed arrow to add samples to the Selected Samples workspace. Click on Next to navigate to the settings and query screen where the default settings are typically used.
3. Hit Next to navigate to the integration method selection screen. Select an appropriate integration method containing MRM transition pairs for lipids to be analyzed. Refer to the Table 1 for MRM transition pairs. Hit Finish.
   NOTE: Retention times will vary depending on instrumentation and individual settings and must therefore be verified for each individual setup.
4. In the navigation bar, click on the Peak Review pane so that MRM pairs can be inspected. To facilitate review, right-click on the Peak Review pane, change the Automatic Zooming to 100% of largest peak, and a Zoom Window of 1.00 - 2.00 minutes.
5. Right-click on the Quantitation Display window and select Analyte and the desired sphingolipid to be examined. Verify that there are no peaks in the Blank samples and that the correct peak has been integrated in the IS samples.
6. Begin integrating unknown samples. Working one sphingolipid class at a time, inspect the retention time for the MRM pair of the internal standard (i.e., C12:0 ceramide) and ensure that the software has integrated the correct peak.
7. Continue onto the analytes in the same lipid class (i.e., C14:0 ceramide, C16:0-ceramide, etc.), beginning with the shortest chain-length lipid in the class. Verify for each chain-length lipid that the software routine has integrated the correct MRM pair peak.
8. When all the analytes have been integrated, create an electronic spreadsheet for each sphingolipid species analyzed (e.g., ceramides, sphingomyelins, etc.). Label column headers to match the order of the analytes and chain-lengths in the LC-MS/MS quantitation software. Also include a column header for sample ID and sample normalization weights determined in step 3.9.
   NOTE: As previously described¹¹, other common normalization measures include total lipid phosphate content or protein amount.
9. Export or copy the peak areas for all analytes and internal standards to the electronic spreadsheet.
10. Normalize data by dividing the integrated peak area of each chain-length species for a given sphingolipid class by the peak area of its corresponding internal standard. For instance, C14:0 ceramide, C16:0 ceramide, C18:1 ceramide, etc., should each be divided by the C12:0 ceramide internal standard peak area.
11. Convert peak area to moles of lipid recovered by multiplying the normalized peak area (calculated in step 6.10) with the amount of internal standard, in picomoles, that was added to the sample in step 4.3. In this protocol, this amount is 250 pmol.
12. To obtain the relative amount of lipid in each sample, divide the picomoles of each lipid chain-length by the tissue weight determined in step 3.9. This will give units of picomoles of lipid per mg of tissue.

Wyniki

In this protocol, we describe in detail a method to remove OCT from cryo-preserved human tissues and weigh the tissues for analysis by LC-ESI-MS/MS. The materials required for this procedure are listed in Table of Materials. Shown in Figure 1 are results of a typical experiment where 10 human lung adenocarcinoma tumors and 10 normal adjacent tissues were washed to remove OCT and analyzed by LC-ESI-MS/MS. Importantly, as we have previously shown¹¹, the...

Dyskusje

OCT is a common long-term cryo-preservation agent used in biorepositories. However, OCT can result in ion suppression when tissues are analyzed by various mass spectrometry platforms^12,13,14,15, or result in loss of signal when samples are analyzed by LC-ESI-MS/MS¹¹. OCT in cryopreserved tissues can also interfere with tissue normalization methods such as weighing and pr...

Materiały

Name	Company	Catalog Number	Comments
1 mL polypropylene pipette tips	NA	NA	Used to retrieve specimens
1.5 mL polypropylene centrifuge tubes	NA	NA
10 mL Erlenmeyer flask	VWR	89091-116	Used for tube and tissue weighing
AB Sciex Analyst 1.6.2	Sciex	NA	Software to analyze and integrate MS data
Ammonium formate	Fisher Scientific	A11550	For LC mobile phases
Analytical scale	NA	NA	Scale that is accurate to 0.1 mg
Bottle top dispenser	Sartorius	LH-723071	Used for dispensing solvents
C12-Ceramide (d18:1/C12:0); N-(dodecanoyl)-sphing-4-enine	Avanti Polar Lipids	LM2212	Internal standard
C12-glucosylceramide (d18:1/12:0); N-(dodecanoyl)-1-β-glucosyl-sphing-4-eine	Avanti Polar Lipids	LM2511	Internal standard
C12-lactosylceramide (d18:1/12:0); N-(dodecanoyl)-1-ß-lactosyl-sphing-4-ene	Avanti Polar Lipids	LM2512	Internal standard
C12-Sphingomyelin  (d18:1/C12:0), N-(dodecanoyl)-sphing-4-enine-1-phosphocholine	Avanti Polar Lipids	LM2312	Internal standard
CHLOROFORM OMNISOLV 4L	VWR	EM-CX1054-1
ClickSeal Biocontainment Lids	Thermo Scientific	75007309	To prevent biohazard aeresols during centrifugation
Conflikt	Decon Labs	4101	Decontaminant
CTO-20A/20AC Column Oven	Shimadzu	NA	For LC
d17:1-Sphingosine;  (2S,3R,4E)-2-aminoheptadec-4-ene-1,3-diol	Avanti Polar Lipids	LM2000	Internal standard
d17:1-Sphingosine-1-phosphate; heptadecasphing-4-enine-1-phosphate	Avanti Polar Lipids	LM2144	Internal standard
DGU20A5R degasser	Shimadzu	NA
Disposable Culture Tubes 13x100mm	VWR	53283-800	13x100 mm screw top tubes
Heated water bath	NA	NA	For overnight lipid extraction
Homogenizer 150	Fisher Scientific	15-340-167	triturate tissues
Homogenizer Plastic Disposable Generator Probe	Fisher Scientific	15-340-177	for homogenization
Kimwipes	Kimtech	34120	Laboratory grade tissue used to make wicks
Methanol LC-MS Grade 4L	VWR	EM-MX0486-1
Nexera LC-30 AD binary pump system	Shimadzu	NA	For LC-MS
Permanent marker	VWR	52877-310
Phenolic Screw Thread Closure, Kimble Chase (caps for disposable culture tubes)	VWR	89001-502	13x100 mm screw top tube caps
Phosphate bufffered saline	Thermo Scientific	10010023	To retrieve specimens from tubes after washing
Repeater pipette	Eppendorf	4987000118	To dispense LC-MS internal standards
Screw Caps, Blue, Red PTFE/White Silicone	VWR	89239-020	Autoinjector vial caps
Screw Thread Glass Vials with ID Patch	VWR	46610-724	Autoinjector vials
SIL-30AC autoinjector	Shimadzu	NA
SpeedVac	Thermo Scientific	SPD2030P1220	For drying solvents
Supelco 2.1 (i.d.) x 50 mm Ascentis Express C18 column	Sigma Aldrich	581300-U	For LC-MS
Triple Quad 5500+ LC-MS/MS System	Sciex	NA	For LC-ESI-MS/MS
Ultrasonic water bath	Branson	Model 2800	for homogenization and resuspension of extracted and dried lipids
Vortexer	NA	NA	For sOCTrP and resuspending dried lipids
VWR Culture Tubes Disposable Borosilicate Glass	VWR	47729572	13x100 glass culture tubes
Water Hipersolve Chromanorm LC-MS	VWR	BDH83645.400