Human PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare) or Lymphoprep (STEMCELL Technologies) density gradient centrifugation, washed twice in PBS, and resuspended at 106 cells/mL in complete RPMI 1640 (cRPMI) medium (Lonza) containing 10% FBS, 2 mM glutamine, and 100 U/mL each of penicillin and streptomycin (Invitrogen). PBMCs were used fresh or cryopreserved in 10% DMSO in FBS and thawed prior to use. Serum was isolated by centrifugation of serum tubes and saving the supernatant in aliquots which were flash frozen in liquid nitrogen prior to cryopreservation in −80C.
Complete rpmi 1640 crpmi medium
Manufactured by Lonza
Complete RPMI 1640 (cRPMI) medium is a cell culture media formulation designed to support the growth and maintenance of a variety of cell types in vitro. It provides a balanced and optimized combination of essential nutrients, vitamins, and other components required for cell proliferation and survival.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Lymphoprep, by STEMCELL (1 mentions)
Rhil 2, by R&D Systems (1 mentions)
Anti cd3 clone hit3α, by BD (1 mentions)
Anti cd28 clone cd28, by BD (1 mentions)
2 protocols using complete rpmi 1640 crpmi medium
1
Isolation and Cryopreservation of PBMCs
2
Isolation and Activation of Human PBMCs
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Human PBMCs were isolated by Ficoll-Paque PLUS (GE Healthcare) density gradient centrifugation, washed twice in PBS, and resuspended at 106 cells/ml complete RPMI 1640 (cRPMI) medium (Lonza) containing 10% FBS, 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin (Invitrogen). Cells were activated with 1 µg/ml anti-CD3 (clone HIT3α; BD) and 1 µg/ml anti-CD28 (clone CD28.2; BD). After 3 d, activated T cells were washed and then cultured in complete RPMI-1640 medium supplemented with 100 U/ml recombinant human IL-2 (rhIL-2; R&D Systems). Transfection was performed with Amaxa Nucleofection kits (Lonza) for primary cells, using C-terminally 5×-myc-tagged constructs in pcDNA3.1.
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