COPARC Biorepository Methods

Transcription

1 Microbiome Oral Wash Processing (Alcoholics and Controls) Decontaminate all surfaces with a 10% bleach solution or other DNA denaturing product. Prepare all reagents, labels, vials, etc. prior to procedures in a laminar flow hood. All processing must be in a laminar flow hood under DNA-free conditions. Collect, process, and run saline controls in parallel to clinical samples. Oral wash should be kept on ice until processing. Processing should be done as soon as possible, but absolutely within 20 minutes. 1. Transfer the oral wash and saline control to sterile 15mL conical tubes and centrifuge at 4 C, 14,000 x g for 20 minutes. 2. Discard the supernatant and resuspend the pellet in 0.5mL sterile PBS. 3. Transfer to a labeled 2mL clear microcentrifuge tube (Fisher # ). 4. Store at -80 C. Microbiome Bronchial Brushings Processing (Alcoholics and Controls) Decontaminate all surfaces with a 10% bleach solution or other DNA denaturing product. Prepare all reagents, labels, vials, etc. prior to procedures in a laminar flow hood. All processing must be in a laminar flow hood under DNA-free conditions. Collect, process, and run saline controls in parallel to clinical samples. The brushes and saline controls will arrive in the lab in labeled clear sterile 2mL microcentrifuge tubes and will have been vortexed once and placed on ice. 1. Vortex on highest setting for 1 minute. 2. Store at -80 C. Non-microbiome Bronchial Brushings Processing (Alcoholics and Controls) Note: The brush will arrive in the lab in a labeled clear sterile 2mL microcentrifuge tube containing RNAlater (Qiagen #76106) and has been vortexed once and placed on ice. 1. Store the microcentrifuge tube at 4 C for at least 48 hours to allow the RNAlater to penetrate the brush. DO NOT freeze immediately. 2. Store the microcentrifuge tube at 20 C until shipment. Transport for analysis within 2-4 months at ambient temperature, except when the temperature is >90 F, when wet ice should be used. Page 1 of 10

2 Exhaled Breath Condensate Processing (All Patients) Note: The RTube (Respiratory Research, Inc #2501) should be capped and stored on ice until ready to process. It should be processed within 20 minutes after collection. 1. Gently roll the RTube to collect all of the sample off the side of the tube. 2. Remove the caps from both sides of the RTube and pool the sample by using the RTube plunger. Ensure the red up arrow is properly oriented while pushing the RTube down onto the plunger. 3. Aliquot 110μl of samples into Nunc 1.8mL cryotubes with blue cryo caps (Nunc #354879). 4. Store at -80 C. Exhaled Breath Condensate for HPLC The RTube (Respiratory Research, Inc #2501) should be capped and stored on ice until ready to process. If study staff is unable to process sample immediately, R-tubes can be capped and frozen in -80 C freezer immediately after collection and should be processed later the same day. The Brown Lab at Emory University will supply the color coded tubes and the preservation solutions. The tubes with preservation solution should be stored at -20 C and thawed on ice before use. Ship specimens on dry ice once every two months or more frequently if desired to Frank Harris at the Brown Lab. Send an or call first to make sure someone is there to receive the samples. 1. Gently roll the RTube to collect all of the sample off the side of the tube. 2. Remove caps from both sides of the RTube and pool the sample by using the RTube plunger. Ensure the red up arrow is properly oriented while pushing the RTube down onto the plunger. 3. Aliquot 200μl into the blue tube (glutathione HPLC) and immediately place on ice. 4. Aliquot 50μl into the pink tube (urea) and immediately place on ice. 5. (Optional) Aliquot 250μl into the orange tube (isoprostane) and immediately place on ice. 6. (Optional) Aliquot the remaining EBC into the brown tube and immediately place on ice. 7. Vortex all samples to mix well. 8. Store samples at -80 C for no longer than 2 months. Page 2 of 10

3 Blood Processing (All Patients) Note: For blood draws use at least a 21G needle to minimize hemolysis. Also avoid aggressive suction during phlebotomy for this same reason. Blood draw for serum, plasma, and buffy coat collection Centrifuge tubes at 900X g for 10 min at room temperature. Green top (sodium heparin)(bd Vacutainer 4mL #367871, 6mL #367878) - aliquot 0.5mL aliquots of plasma off the top phase into 1.8mL cryotubes (Nunc #375418) with green cap inserts (Nunc #355018), then collect red blood cells (RBCs) from the bottom phase and aliquot them into 1.8mL cryotubes with yellow cap inserts (Nunc #355077). A 4mL green top will typically yield between 1 and 3mL of plasma. Red top (BD Vacutainer 4ml #367812, 6mL #367815, 10mL #367820) aliquot 0.5mL aliquots of serum off of the top phase into 1.8mL cryotubes with red cap inserts (Nunc #354968). A 6mL red top will typically yield between 1 and 4mL of serum. Purple top (EDTA)(BD Vacutainer 4mL #367861, 6mL #367863) aliquot 0.5mL aliquots of plasma off the top phase into 1.8mL cryotubes with purple cap inserts (Nunc #375922). A 4mL purple top will typically yield between 1 and 3mL of plasma. Then collect the buffy coat by pipetting the white layer of cells between the top phase and the red blood cell (RBC) bottom layer and pipetting it into a 1.8mL cryotube with a pink cap insert (Nunc #375884). Light Blue Top (Na Citrate) (BD Vacutainer 4.5mL #363083) aliquot 100μl aliquots into 1.8mL cryotubes with white cap inserts (Nunc #354755). Optional: Add 10uL of 100uM BHT (from McCord Lab) per ml of blood to EDTA plasma, sodium heparin, plasma, and serum for TBARS and Pon1 assays. Blood draw for HPLC measurements 1. Draw blood into syringe and immediately add 1 ml of whole blood to 500uL of blood preservation solution (from Brown lab) in a clear 1.5mL microcentrifuge tube (Fisher Scientific # ). Note: Use at least a 21G needle to minimize hemolysis. 2. Centrifuge for 10 min at 500 X g 3. Aliquot 250uL of the upper phase into a green 1.5mL microcentrifuge tube (Fisher Scientific # ) containing 250uL GSH preservation solution (from Brown lab). 4. Store at -80 C. Page 3 of 10

4 BAL Processing (Microbiome Alcoholics and Controls) Decontaminate all surfaces with a 10% bleach solution or other DNA denaturing product. Prepare all reagents, labels, vials, etc. prior to procedures in a laminar flow hood. All processing must be in a laminar flow hood under DNA-free conditions. 1. Prior to centrifugation for isolation of alveolar cells (see below), remove 2mL of Lower BAL and place into a clear 2mL microcentrifuge tube (Fisher# ). Centrifuge at 25 X g for 1 minute to settle the cells. 2. Without disturbing the cell pellet, very gently pipette out 1.5mL of the cell-free lavage and transfer to a labeled clear 2mL microcentrifuge tube. 3. Centrifuge at 14,000 X g for 20 minutes. Decant off supernatant. 4. Store the pellet at -80 C. BAL Processing (Alcoholics and Controls) In general, mL sterile isotonic saline will be used for bronchoalveolar lavage, instilled in 50mL aliquots. We have used hand aspiration to more carefully control suction amount in the lung. The first 50mL aliquot instilled/aspirated is reflective of the airways, and is kept separate from the subsequent aliquots and should be marked Upper. The volume aspirated from this first aliquot (assuming 50mL instilled) is generally only 10-20mL. The second, third, and any subsequent aliquots reflect distal airway/alveolar sampling and should be marked Lower. Aliquot cell free BAL in a biosafety cabinet (hood). 1. Record volume received. 2. Centrifuge the 50mL conical tubes of BAL fluid 900 x g for 10 min at room temperature. 3. Place samples on ice while aliquotting. BAL for HPLC Measurements: For Upper and Lower add 500uL BAL and 100uL GSH preservation solution (from Brown lab) to a yellow 1.5mL microcentrifuge tube (Fisher Scientific # ), add 500uL BAL and 500uL GSH preservation solution to a blue 1.5mL microcentrifuge tube (Fisher Scientific # ), and add 1.5mL BAL to an amber 1.5mL microcentrifuge tube (Fisher Scientific # ). There should be one set of tubes for the Upper samples and one set of tubes for the Lower samples. Optional: For Lower BAL, add 10uL of 100uM BHT (from McCord lab) per ml of BAL to 4mLs of BAL and aliquot into 1.8mL cryotubes (Nunc #375418) with orange cap inserts (Nunc# ) for TBARS and Pon1 assays. 4. Aliquot remaining BAL in 3-3.5mL aliquots in sterile 5mL screw top tubes (autoclaved Sarstedt # and # or Nunc #348100). 5. Store all BAL samples at -80 C. Page 4 of 10

5 BAL Cell Processing 1. In a biosafety cabinet (hood), resuspend the cells from the above aliquots ( Upper and Lower ) in a total of 10mL 10mL RPMI (Cellgro # CV) and antibiotics (Cellgro #MT CI). Place 2-3mL of media in each 50mL conical tube, gently resuspend the cells, and then combine all of the resuspended cells into one tube. 2. Count cells and check viability by diluting 10μl of sample with 90μl trypan blue (VWR # ) and applying 10μl of the dilution to a hemacytometer. Count all of the cells in the 4 quadrants then divide that number by 4. Multiply that number by 10 4 to calculate how many cells there are per ml. Multiply that number by the total volume (10mL) to calculate the total number of cells in the 10mL of cell suspension. An automated cell counter can also be used, if available. 3. Divide up the cells according to assay need below. Cytospins: Resuspend 120,000 macrophages in 400ul of 1XDPBS (Cellgro # CV). Cytospin 4 slides (Fisher Scientific # ) at 30,000 cells per slide, following the equipment s instructions. Immediately after cytospin, fix 2 of the slides in 2% paraformaldehyde (Electron Microscopy Sciences #15710, diluted in 1XDPBS) for 15 minutes at room temperature. Immediately place slides in 70% EtOH for 1 minute, remove and store in 1XDPBS at 4 C. The remaining 2 slides are stained using Protocol Hema 3 (Fisher Scientific # ), dried, and stored in a slide box at room temperature for subsequent tabulation of differentials. Cells for RNA: 1-2x10 6 of cells are centrifuged at 900 X g for 5 minutes to pellet the cells. The media is aspirated off and discarded, and then the cells are immediately frozen and stored at -80 C. Cells for proteins: 1-4x10 6 of cells are centrifuged at 900 X g for 5 minutes to pellet the cells. The media is aspirated off and discarded, then the cells immediately frozen and stored at -80 C. Freeze down protocol for cryostorage (For BAL cells and PBMCs): Pellet cells by centrifuging them at 900 X g for 5 minutes. Resuspend them with room temperature DMEM freezing media (35mL DMEM-Cellgro # CV, 10mL FBS-Atlanta Biologicals #S11550H, 5mL DMSO- Sigma #D2650, filter sterilized -Corning #430320) at a final concentration of 2-4 x10 6, aliquot into 1.8mL cryotubes (Nunc #375418), and place on ice. Place them in a freezing container (Nalgene Cryo 1 C Freezing Container or Biocision Cool Cell) and store at -80 C for 24 hours. Store the cells in a liquid nitrogen storage tank long term. Page 5 of 10

6 Secretion of cytokines/growth factors by treated cultured Alveolar Macrophages (AMs) in media: 1. Plate 500,000 macrophages in 2mL RPMI (Cellgro # CV) and antibiotics (Cellgro #MT CI) per well in a 12 well plate (Costar #3513), incubate at 5% CO 2 and 37 C for at least 1 hour to allow the cells to adhere. 2. Dilute 1mg/mL stock LPS (Sigma #L-6529) in 10mL RPMI with antibiotics to a concentration of 2ug/mL. 3. Dilute 1mg/mL stock LTA (InvivoGen #tlrl-pslta) in RPMI with antibiotics to a concentration of 10ug/mL. 4. Dilute 1M NAC (Sigma #A9165) in RPMI with antibiotics to 5mM. 5. AMs only: For two of the macrophage plated wells, aspirate the media and replace with 2 ml of fresh RPMI with antibiotics. 6. AMs + LPS: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and LPS and 1mL of RPMI with antibiotics for a final LPS concentration of 1ug/mL. 7. AMs + LTA: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and LTA and 1mL of RPMI with antibiotics for a final LPS concentration of 5ug/mL. 8. AMs + NAC: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and NAC and 1mL of RPMI with antibiotics for a final NAC concentration of 2.5mM. 9. AMs + LPS + NAC: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and NAC and 1mL of RPMI with antibiotics and LPS. 10. AMs + LTA + NAC: For two of the macrophage plated wells, aspirate the media and replace with 1mL of fresh RPMI with antibiotics and NAC and 1mL of RPMI with antibiotics and LTA. 11. Incubate the plate O/N at 5% CO 2 and 37 C. 12. The next day, collect the media in 1mL aliquots, 2 aliquots per well and store at - 80 C. Well 1 aliquots should be labeled 1A and 1B and well 2 aliquots should be labeled 2A and 2B. 13. Save the cells for RNA extraction by adding 350μl Buffer RLT from an RNeasy Mini Kit (Qiagen #74104) with β-mercaptoethanol (Sigma #63689) to each well in order to lyse the cells. Scrape the cells, collect all of the Buffer RLT and cells from each condition and load the collected sample onto a QiaShredder column (Qiagen #79654) placed inside a 2mL collection tube from the kit. Centrifuge at max speed for 2 minutes. Discard Qiashredder column and cap the tube with sample with the provided caps and store the homogenized sample at -80 C for up to 3 months. Page 6 of 10

7 BAL and BAL Cell Processing (Burn Patients) BAL will be immediately transported on ice to the lab. Aliquot BAL in a biosafety cabinet (hood). Wash buffer PBS(Gibco #14190) with 5% FBS (Gibco #16000, certified US origin) and 1% PSG(Gibco #10378) Media - RPMI (Gibco #21870) with 5% FBS (Gibco #16000, certified US origin) and 1% PSG(Gibco #10378) 1. Record volume received. 2. Add a volume of wash buffer (see above) equal to 50% of the total volume of the sample. For example, if the sample is 10mL, add 5mL of wash buffer. 3. Filter the raw sample through a BD Falcon 100 micron cell strainer (BD #352360) into a 50mL conical tube (BD Falcon #352098). The sample only needs to be filtered once, but more than 1 filter may be necessary due to the particulate matter or mucous in the sample. 4. Centrifuge at 1200 rpm for 5 minutes. 5. Aliquot supernatant in 300μl aliquots into orange microcentrifuge tubes (Fisher # ) and store at -80 C. 6. Resuspend the cell pellet in 5mL of wash buffer with 0.5mg DNase (Roche # ) and 5mg MgCl (Fisher #BP ). 7. Incubate at 37 C, 5% CO 2 for 10 minutes. 8. Centrifuge at 1200 rpm for 5 minutes. 9. Resuspend pellet in 5mL ACK RBC lysis buffer (Gibco #A10492). 10. Incubate at room temperature for 2 minutes then add 10mL of wash buffer. 11. Centrifuge at 1200 rpm for 5 minutes. 12. Resuspend the cells in 5mL of media (see above) 13. Count cells and check viability by diluting 10μl of sample with 90μl trypan blue (VWR # ) and applying 10μl of the dilution to a hemacytometer. Count all of the cells in the 4 quadrants then divide that number by 4. Multiply that number by 10 4 to calculate how many cells there are per ml. Multiply that number by the total volume (5mL) to calculate the total number of cells in the 5mL of cell suspension. An automated cell counter can also be used, if available. 14. Adjust cell concentration to 1X10 6 cells/ml in media. 15. Create two cytospins using 100μl of cell suspension per slide, following the equipment manufacturer s instructions. The slides are stained using Protocol Hema 3 (Fisher Scientific # ), dried, and stored in a slide box at room temperature for subsequent tabulation of differentials. 16. Plate the remaining cells in a flat bottomed 96 well culture plate (Costar #3596), 100μl per well. There will typically be enough cell suspension to plate between 4 and 20 wells. 17. For half of the wells, add LPS (Sigma #L-6529) for a final concentration of 100ng/mL. 10μl aliquots of 1 mg/ml LPS should be diluted with 100μl of media and 1μl used per well to bring the final concentration in the well to 100ng/mL. 18. Incubate the plate at 37 C, 5% CO 2 for 20 hours. 19. Centrifuge plates at 1085 rpm for 5 minutes. Harvest the supernatants by type (LPS treated or non-treated), vortex, and aliquot 110μl in purple microcentrifuge tubes (Fisher # ). 20. Store at -80 C. Page 7 of 10

8 Additional protocols for saved samples RNA Extraction from cell pellets: Frozen cell pellets are used for RNA extraction using the RNeasy Mini Kit (Qiagen #74104), following the standard protocol with optional QiaShredder columns (Qiagen #79654). RNA quantity and quality is determined using the NanoDrop Spectrophotometer (Thermo Scientific). RNA Extraction from bronchial brushings: Frozen cell pellets are used for RNA extraction using the RNeasy Micro Kit (Qiagen #74004), following the standard protocol. RNA quantity and quality is determined using the NanoDrop Spectrophotometer (Thermo Scientific). DNA Extraction from bronchial brushings: Frozen cell pellets are used for DNA extraction using the QIAamp DNA Mini Kit (Qiagen #51304), following the standard protocol, as well as Appendix E and Appendix D instructions. 1. Centrifuge the sample for 10 minutes at 5000 x g (7500 rpm). 2. Resuspend the pellet in 600μl sorbitol buffer (Sigma #S6021). 3. Add 200 U lyticase (Sigma #L2524) and incubate in a heat block set at 30 C for 30 minutes. 4. Centrifuge for 10 minutes at 5000 x g (7500 rpm). 5. Resuspend the pellet in 180μl of 20 mg/ml lysozyme (Sigma #L7651); 20 mm Tris-HCl ph 8.0 (Invitrogen # ); 2 mm EDTA (Sigma #E6758); 1.2% Triton (Fisher #327371). 6. Incubate at least 30 minutes at 37 C. 7. Add 20μl proteinase K and 200μl Buffer AL and mix by vortexing. 8. Incubate at 56 C for 30 minutes. Vortex every 10 minutes to increase yield. 9. Centrifuge for a few seconds. 10. Follow the Protocol: DNA Purification from Tissues from step 6 (page 34). DNA quantity and quality is determined using the NanoDrop Spectrophotometer (Thermo Scientific). Protein extraction: Proteins from cultured and uncultured cells are extracted using the M-PER Mammalian Protein Extraction Reagent (Pierce #78503). Each ml of the M-PER is supplemented with 10μl of a 1:100 dilution of protease inhibitor (Sigma #P8340), 2μl of 100mM PMSF (Sigma #P7626), and 5μl of 100mM DTT (Sigma #D5545). Thaw and resuspend the frozen cell pellets in 20μl of supplemented M-PER per 1X10 6 cells. Rotate the resuspended cells end over end using a tabletop rotator for 15 minutes at room temperature. Centrifuge the samples at 13,000 X g for 15 minutes at 4 C to pellet the cell debris. Aliquot the supernatants containing the protein lysates in 20μl volumes in 0.5mL microcentrifuge tubes (Fisher Scientific # ) and stored at -80 C. Bradford assay for protein measurements: Total protein for the protein lysates and for the cell free BAL are measured using the Coomassie (Bradford) Protein Assay Kit (Pierce #23200). Page 8 of 10

9 Optional protocols for fresh cells Alveolar Macrophage (AM) Apoptosis: Aliquot 250,000 alveolar macrophages for each tube into three 5mL pop-top tubes (Falcon #352058) and bring the volume of each tube up to 500μL with DMEM (Cellgro # CV) with 10% FBS (Atlanta Biologicals #S11550H) and antibiotics (Cellgro #MT CI). Incubate the tubes for 1 hour at 37 C in an incubator shaker set at 200rpm.Thaw and dilute an aliquot of TNF-α (GeneScript #Z01001) with 90μl of 1XDPBS (Cellgro # CV) for a final concentration of 1ng/μl. Add 20μl of the TNF-α to one tube (40ng/mL TNF-alpha), and 40μl of the TNF-α to another tube (80ng/mL TNF-α). The remaining tube will be an alveolar macrophage only control. Incubate 2 hours at 37 C in an incubator shaker set at 200rpm. Mix cells gently with a pipette and cytospin 250μL per slide (Fisher Scientific # ), two slides per treatment. Immediately after cytospin, fix in 2% paraformaldehyde (Electron Microscopy Sciences #15710, diluted in 1XDPBS) for 15 minutes at room temperature. Immediately place slides in 70% EtOH for 1 minute, remove and store in 1XDPBS at room temperature. The slides were then used in conjunction with the DeadEnd Fluorometric TUNEL System (Promega #G3250). Cells for overnight culture: Cells are plated in DMEM (Cellgro # CV) with 10% FBS (Atlanta Biologicals #S11550H) and antibiotics (Cellgro #MT CI) at a concentration of 500,000 cells per ml of media in a 6 well plate (Costar #3516). Incubate at least 1 hour at 5% CO 2 and 37 C to allow the cells to adhere. Aspirate the media off the cells and replace with fresh media. Culture the plate overnight at 5% CO 2 and 37 C. The cells are then scraped in the plate and the media with cells is pipetted into a 15mL conical tube and centrifuged at 900 X g for 5 minutes. The media is aspirated off and aliquotted into 1.5mL microcentrifuge tubes (Fisher Scientific # ). The media and the pellet in the 15mL conical tube are stored at -80 C. Alveolar Macrophage (AM) phagocytosis: For twelve wells of a flat bottomed 96-well plate (Costar #3596), add 100,000 cells in DMEM (Cellgro # CV) with 10% FBS (Atlanta Biologicals #S11550H) and antibiotics (Cellgro #MT CI) to each well and allow to adhere for at least 1 hour at 5% CO 2 and 37 C. At the end of the day, change media and add 80ng/mL of TNF-α (GeneScript #Z01001) in 1XDPBS (Cellgro # CV) to 4 wells, with the same volume of 1XDPBS added to the remaining 8 wells. Incubate the plate overnight at 5% CO 2 and 37 C. The next morning use the plate with the Cytoselect 96-Well Phagocytosis Assay Zymosan, Colorimetric Format (Cell Biolabs Inc #CBA-224). At the beginning of the assay, add zymosan from the kit to 2 wells of the untreated cells for 1 hour, add zymosan to 2 wells of the untreated cells for 2 hours, add zymosan to 2 wells of the TNF-α treated cells for 1 hour, add zymosan to 2 wells of the TNF-α treated cells for 2 hours, and add 1XDPBS to 4 wells as a control for 2 hours. After the fixation step, fill 2 of the control wells with 200μl of 1XDPBS and after the rest of the protocol is finished, stain those 2 wells using Protocol Hema 3 (Fisher Scientific # ) take pictures. The remaining wells follow the protocol as written. Page 9 of 10

10 Secretion of cytokines/growth factors by cultured Alveolar Macrophages (AMs) in media: Resuspend Jurkat cells (ATCC) at 5x10 6 cells/ml in RPMI (Cellgro # CV) with 10% FBS (Atlanta Biologicals #S11550H) and antibiotics (Cellgro #MT CI). Place the cells under UV light for 10 minutes, and then incubate at 5% CO 2 and 37 C for 3 hours. Plate 250,000 macrophages per well in 8 wells of a 24 well plate (Costar #3524), incubate at 5% CO 2 and 37 C for at least 1 hour to allow the cells to adhere. Pellet Jurkat cells, resuspend them in 1 ml DMEM (Cellgro # CV) with 10% FBS (Atlanta Biologicals #S11550H) and antibiotics (Cellgro #MT CI). Recount the cells and adjust cell concentration to 5x10 6 cells/ml. Dilute LPS (List Biological Laboratories Inc) in DMEM with FBS, and antibiotics to a concentration of 2ug/mL. AMs only: For two of the macrophage plated wells, aspirate the media and replace with 1 ml of fresh DMEM with FBS, and antibiotics. AMs + LPS: For two of the macrophage plated wells, aspirate the media and replace with 500ul of fresh DMEM with FBS, and antibiotics and 500ul of the LPS in media, for a final LPS concentration of 1ug/mL. AMs + Apoptotic Jurkats: For the third set of wells, aspirate the media and replace it with 500ul of fresh DMEM with FBS, and antibiotics and 500ul of the UV treated Jurkats in DMEM, for a final Jurkat cell concentration of 2.5 x10 6 cells/ml. AMs + Apoptotic Jurkats + LPS: For the final set of wells, aspirate the media and replace it with 500ul of the LPS in media and 500ul of the UV treated Jurkats in DMEM, with FBS, and antibiotics for a final LPS concentration of 1ug/mL and a final Jurkat cell concentration of 2.5 x10 6 cells/ml. Incubate the plate O/N at 5% CO 2 and 37 C. The next day, collect the media, aliquot in 500ul aliquots and store at -80 C. Optional: Save the cells for protein extraction by scraping the cells and collecting the cells and media in a 15mL conical tube. Centrifuge at 900 X g for 5 minutes. Aspirate and aliquot the media in 500ul aliquots in fresh microcentrifuge tubes (Fisher Scientific # ). Store pellets and media at -80 C. Page 10 of 10