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Bone Marrow EPC Isolation.doc

2 Pages

Course Code
BIOL 2020
Hernan Humana

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-[ Protocol for Cell Labeling and Injection ]- Cell labeling (CMTMR/ CMFDA) and preparation of cells for injection Day 0 1. Place EGM-2MV, EBM, and PBS in water bath 2. Prepare staining solution: − Reconstitute CMTMR/CMFDA (50ug) with 1mL DMSO. − For each flask: 90 uL of CMFDA solution is needed for each 5mL of EBM − Master staining mix: In a 50 ml Falcon tube = Add 90 uL CMFDA x n of flask you have and 5ml x n of flask you have (the resulting CMFDA concentration in the 5 mL is 20 mM) − Ex 7 flask= 90 X 7 = 630 uL; 5 ml EBM x 7 = 35ml 3. Wash with PBS (10mL each flask), aspirating old media out of flask. 4. Aspirate PBS. 5. Put 5mL of Staining solution mix into each flask. Incubate in incubator for 45min. 6. Wash with PBS (aspirate the staining solution). Add 10mL EGM-2MV. 7. Place in incubator at least 24hr. In summary: Wash  Stain  Wash  Medium Day 1 1. Place EGM-2MV, trypsin, and PBS in water bath 2. Wash with PBS (10mL each flask) 3. Aspirate the PBS. Add 3mL Trypsin to each flask. Incubate for 5min in incubator. Note 1: to do in batches of 4 Note 2: It is empirical that the flasks get 5min. Any longer will kill the cells! 4. Take flasks out of incubator, check under microscope (For Microscope dummy user: yes, place the flask under the lens). The cells should be floating together. 5. Smack the flask forcefully 4 times. 6. Add 7mL EGM-2MV, to deactivate trypsin. Use 25mL pipette to do it all flasks at once. 7. Transfer the content of each flask into 15mL falcon tube. So, there should be 7 falcon tubes. 8. Centrifuge at 360 RCF, 10min 9. Aspirate supernatant in each falcon tube 10. Combine all 7 tubes and re-suspend with 7mL of PBS. Re-suspend well, avoid clumping. 11. Take 20uL of cells, add to 20mL of Iso Sheath Fluid in a cuvette. Flow: W
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