B. Separation of peripheral blood mononuclear cells (PBMC)
1.Fill in all required information on data sheet “Cryopreservation of Peripheral Blood Mononuclear Cells.”
2.Remove the rubber stoppers from the vacutainer tubes. Wipe off the top of each tube with an alcohol swab.
3.Transfer the blood to an appropriate size flask using a sterile pipet. Dilute the blood 1:1 with RPMI 1640. Use approximately 10 ml RPMI 1640 to rinse the vacutainer tubes, and combine with the rest of the blood.
4.Transfer 13 ml Ficoll (warmed to room temperature) to each 50 ml centrifuge tube needed to process the entire sample.
5.Carefully layer the diluted blood over the Ficoll.
6.Centrifuge at 2000 rpm for 15 minutes with centrifuge brake OFF.
7.Collect the cell band at the interface from each centrifuge tube, and transfer it to fresh centrifuge tube. Add RPMI 1640 for a final volume of 45 ml in each centrifuge tube. Centrifuge at 2000 rpm for 5 minutes. Remove the supernatant, combine the cells in all of the tubes into one tube in 45 ml RPMI 1640. Count the cells.
C. Cryo-preservation of isolated PBMC
1.Centrifuge cells at 2000 rpm for 5 minutes. Remove the supernatant.
2.Resuspend the cells at 20 x 106 cells/ml in freezing solution (50% FCS, 40% RPMI 1640, and 10% DMSO).
3.Transfer 1 ml of cells into each 1.8 ml cryovial that is labeled with donor’s name, hospital number, date, and cell type.
4.Transfer all cryovials to a pre-cooled freezing chamber and freeze using the appropriate program.
5.Once the program is complete, transfer the cryovials to a liquid nitrogen freezer作者: fox_79 时间: 2012-4-13 16:32