Cryopreserved vial (10 x 10^6 cells) of CD8+ T cells that were negatively selected from freshly isolated primary human peripheral blood mononuclear cells (PBMCs). The PBMCs came from a healthy donor and were isolated from whole blood or leukapheresis samples using a Ficoll gradient. Magnetic antibodies to monocytes, granulocytes, CD4+ T cells, gamma/delta T cells and other immune subsets present in PBMCs were then used to purify untouched CD8+ T cells via immunomagnetic separation. Before and after CD8+ T cell isolation, the cells were stained to evaluate purity and viability by flow cytometry. Cells were cryopreserved in CryoStor CS10 cryopreservation medium (Stemcell #07930) at a controlled rate.
Source Normal human PBMC from Leukapheresis Sample
Store cells at -135°C or colder. Thawed cells should be used immediately for downstream applications. Because these are primary cells, we do not recommend maintaining these cells in culture for long periods of time.
Growth Media
For best results, it is highly recommended to use these validated and optimized media from BPS Bioscience. Other preparations or formulations of media may result in suboptimal performance.
TCellM™ (BPS Bioscience #78753): Contains Iscove’s MDM, Heat inactivated Fetal Bovine Serum (HiFBS), 1% penicillin/streptomycin, 2-Mercaptoethanol, insulin, and other supplements
Instructions for Use
First, pre-warm Thaw Medium 10 to 37°C. It is important to work quickly in the following steps to ensure high cell viability and recovery. Quickly thaw cells in a 37°C water bath with constant but gentle agitation. Clean the outside of the vial with 70% ethanol and immediately transfer the entire contents to a 50 ml tube. Slowly add 10 ml of pre-warmed medium while gently swirling the tube to mix. Centrifuge the cell suspension at 300 x g for 15 minutes at room temperature. Carefully remove the supernatant with a pipette without disturbing the pellet. Gently resuspend the cell pellet by flicking the tube, then add the desired volume of warm medium, and mix. NOTE: Up to 30% cell loss can be expected during washing steps. Cells are now ready for use in downstream applications.
Shipping Temperature
-80°C
Notes
Characterization Criteria Cell count, viability (trypan blue exclusion and FACS with impermeable DNA binding dye), and surface expression of CD3 and CD8
Warnings
Donors have been screened and determined negative for:
Hepatitis B (anti-HBc EIA, HBsAg EIA)
Hepatitis C (anti-HCV EIA)
Human Immunodeficiency Virus (HIV-1/HIV-2 plus O)
Human T-Lymphotropic Virus (HTLV-I/II)
HIV-1/HCV/HBV
West Nile Virus
Trypanasoma cruzi
Note: Testing cannot guarantee that any sample is completely virus-free. These cells should be treated as potentially infectious and appropriate biological safety level 2 precautions should be used.
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