Cell culture and cell isolation protocols
Lymphocyte Harvest and T Cell Isolation from Human Donors
After determination that the donor is HLA-matched with recipient, the donor undergoes a 2 to 5 liter apheresis procedure using a CS-3000 or an equivalent machine. The apheresis product is subjected to counterflow centrifugal elutriation by standard operating procedures of the NIH Department of Transfusion Medicine, Cell Processing Section. The lymphocyte fraction of the elutriation product (120 to 140 fraction) is depleted of B cells by incubation with an anti-B cell antibody (anti-CD20; Nexell) and an anti-CD8 antibody (Nexell) and sheep anti-mouse magnetic beads (Dynal; obtained through Nexell) by standard operating procedures using the MaxCep Device (Nexell). Flow cytometry will be performed to document that CD8 + T cell contamination is <1%. The resultant CD4-enriched donor lymphocyte product can be cryopreserved in aliquots of 50 to 200times10 6 cells/vial. Sterility of the population is not tested at this early stage of the Th2 cell generation procedure; such testing occurs after final co-culture of donor CD4 cells.
Disclaimer: Sciclips does not guarantee the authenticity or validity of the protocols listed in this section. These protocols are extracted "as is" from various sources, with minor modifications. Please refer to the respective citations, which are hyperlinked, for more details. The use of cited molecules, not limited to proteins, nucleic acids, compounds etc, in the protocols that are extracted from patents may be covered under patent rights. These molecules should be used for information purpose only.
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