Bronco Alveolar Lavage (BAL) Processing A. Reagents Needed RNA Later Solution PBS Sterile solution 1X B. Objective: to separate and store fluid and pellet from BAL. C. Description 1. Use 40m cell strainer to remove debris. 2. Centrifuge BAL sample at 1500RPM for 5 minutes at room temperature (RT). 3. Harvest supernatant (fluid) into a separate 15ml falcon tube. 4. Reserve pellet. ` BAL Fluid 5. Aliquot supernatant into 5 labeled cryovials (up to 1.5ml/vial) and store at ‐80°C. BAL Pellet 6. Re‐suspend the pellet in the first tube in 10ml PBS at RT. 7. Centrifuge at 1500 rpm for 5 minutes at RT. 8. Re‐suspend pellet in 600 l in RNA Later solution and store in 2 eppendorf tubes. Blood Processing A. Reagents Needed DNAzol PBS Sterile solution 1X Ficoll ACK Lysing Buffer Trypan Blue Chilled Filtered 0.22m 100% FCS Chilled Filtered 0.22m 80% FCS 20% DMSO B. Objective: to separate and store DNA, Blood Plasma and PBMCs. C. Description DNA 1. Invert the blood tube 2‐3 times to ensure homogenous mixture. 2. Using a sterile pipet, take out 1ml of whole blood and transfer into 15ml falcon tube. 3. Mix whole blood with 2ml of DNAzol. 4. Aliquot Blood‐DNAzol mixture (1.5ml/vial) into two labeled cryovials. 5. Store vials immediately at ‐80° C. Plasma 1. Centrifuge blood samples at 1500RPM for 10 minutes at Room temperature (RT). 2. Reserve spun blood. 3. Harvest supernatant (Plasma) into separate 15ml falcon tube. 4. Re‐spin the plasma at 2500RPM for 5 minutes at RT. 5. Aliquot supernatant into 5 labeled round‐bottom cryovials and store in ‐80° C Isolating PBMC 6. Add 5ml of Ficoll to each 15ml tube (or 10 of Ficoll to the 50ml Falcon tube). 7. Re‐suspend the whole blood to two times its original volume with sterile PBS. 8. Homogenize by pipetting up and down. 9. Use a 15ml Falcon tube if the amount of blood is below 4.5ml, if above use 50ml tubes. 10. Tip tube of Ficoll almost horizontally and add diluted blood EXTREMELY SLOWLY right above the Ficoll. Do not let the blood and Ficoll mix, Ficoll and blood layers should remain separate. 11. Centrifuge the Ficoll/blood at 2500RPM for 15 minutes at RT. IMPORTANT: set deceleration to “0” and acceleration to “3” 12. Harvest the cell ring (PBMC) located above the Ficoll in a circular movement. 13. Transfer cells into a second 15ml or 50ml tube filled with PBS (5ml for 15ml tube/15ml for 50ml tube). 14. Spin cells at 1500RPM for 5 minutes (spin 10min if using 50ml tube) at RT. 15. Discard supernatant and gently tap bottom of tube to re‐suspend pellet. 16. Re‐suspend cells again with PBS 1X (if pellet looks red, re‐suspend cells in 3‐5ml of ACK buffer to lyse remaining RBC’s, wash with PBS after 5 minutes) Transfer the cell suspension into a 15ml tube, if originally in 50ml tube. 17. Spin cells again at 1500RPM for 5 min at RT. 18. Discard supernatant, re‐suspend in 5ml PBS. Cell Counting 19. Pipet cell suspension up and down to homogenize. Take a drop from the center. 20. Pipette 10l of Trypan Blue into a test tube, add 10l of cell suspension and mix thoroughly. Add 10l of cell/dye mixture to hemocytometer. 21. Count 2 quadrants of cells, take the average, and apply cell concentration formula: #of cells x 2 x total volume of cell suspension x 10^4 (e.g. 40 cells counted in 5ml of PBS→ 40 x 2 x 5 x 10^4 = 4 x 10^6 cells total). Count at least 50 cells, if less than 50 cells in 2 quadrants, count all 4 quadrants and average. Cryopreservation 22. Determine number of round‐bottom cryovials to make as follows:  Less than 3 x 10^6 cells: 2 cryovials.  3‐4 x 10^6 cells: 3 cryovials.  4‐5 x 10^6 cells: 4 cryovials.  Continue up to 5 cryovials max post‐transplant; 15 max pre‐
transplant (amount may vary depending on study/organ type). 23. FREEZE ONLY 10 or less aliquots at a time. 24. Always keep 0.22m filtered FCS and FCS/DMSO in ice. 25. After counting cells, centrifuge cell suspension at 1500RPM for 5 minutes at RT. 26. Label appropriate number of cryovials and chill on ice. 27. Discard supernatant and re‐suspend cells in 0.22m filtered FCS (0.5ml/vial to be made). 28. Distribute 0.5ml of cell re‐suspended in ice‐cold FCS into each chilled cryovial. 29. Add 0.5ml ice cold 0.22m filtered 80% FCS/ 20% DMSO to each cryovial of cells to make a 1:1 solution of FCS/FCS‐DMSO 30. Recap and turn vial containing cells and preservative upside down several times to homogenize and quickly store in ‐80° C. 31. Long‐term storage of frozen cells in Li N2. Serum Processing A. Objective: to isolate serum from whole blood. B. Description 1. Collect blood in Serum Separating tubes (SST). 2. Let it sit for 30‐45 minutes to coagulate. 3. Spin tubes at 3500 rpm for 15 minutes at room temperature. 4. Collect the layer above the filter and aliquot needed amount into vials CCTI Sample Repository Standard Operating Procedure
Urine Processing A. Reagents Needed PBS 1X sterile RNA Later RNaseZap B. Objective: to separate and store urine and urine pellet. C. Description 1. Distribute urine into 15‐50ml or conical “football” tube (label with subject name). 2. Spin urine at 3000RPM for 30 minutes at 4° C. 3. Aliquot supernatant (without pellet) into 5 (max) round‐bottom cryovials‐ labeled with barcode (1.5ml per vial). 4. Store aliquots at ‐80° C. 5. Discard the remaining supernatant and wash the urine pellet with 10ml sterile PBS. 6. Spin pellet at 3000RPM for 15 minutes at 4° C. 7. While the pellet is spinning, clean hood bench and surrounding areas (includes pipetter, pipetman) with RNaseZap to remove Rnase. 8. Label 2 RNAse free orange‐top cryotubes with barcode. 9. Re‐suspend urine pellet in 600l of RNA later. 10. Aliquot re‐suspended urine pellet into the 2 labeled tubes (300l/tube). 11. Store at ‐80° C.