Purified naive and total human CD8+ T cells were cultured in RPMI 1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, respectively. Cells were seeded at 5 to 10×104 cells into 96-well U bottom plate and stimulated with anti-CD3/CD28 coated microbeads (Dynabeads® T-Activator CD3/CD28; Thermo Fisher Scientific, Waltham, MA, USA) in the absence or presence of the indicated cytokines; recombinant human (rh) IL-1β (25 ng/ml), rhIL-6 (6.25 to 100 ng/ml), rhIL-21 (0.1 to 100 ng/ml), rhIL-7 (50 ng/ml), IFN-α (50 or 100 ng/ml), rhIL-10 (6.25 to 100 ng/ml; all from R&D systems, Minneapolis, MN, USA), rhIL-12 (50 ng/ml), rhIL-15 (50 or 100 ng/ml), rhIL-2 (100 IU/ml, all from PeproTech, Rocky Hill, NJ, USA). In some experiments, cells were stimulated with anti-CD3/CD28 coated microbeads with chemical inhibitors: STAT1 inhibitor Fludarabin, STAT3 inhibitor 5,15-DPP, or STAT5 inhibitor CAS 285986-31-4 (all from Sigma-aldrich, St. Louis, MO, USA) .
Rhil 21
Manufactured by R&D Systems
Sourced in United States
RhIL-21 is a recombinant human interleukin-21 protein. Interleukin-21 is a cytokine that plays a role in the regulation of the immune response.
Automatically generated - may contain errors
Lab products found in correlation
Rhil 2, by Thermo Fisher Scientific (1 mentions)
Rhil 7, by R&D Systems (1 mentions)
Rhil 15, by Thermo Fisher Scientific (1 mentions)
Rhil 12, by Thermo Fisher Scientific (1 mentions)
Rhil 6, by R&D Systems (1 mentions)
Rhil 10, by R&D Systems (1 mentions)
Ifn α, by R&D Systems (1 mentions)
Cpg odn 2006, by InvivoGen (1 mentions)
Human b cell isolation kit 2, by Miltenyi Biotec (1 mentions)
2 protocols using rhil 21
1
Modulation of Human CD8+ T Cell Activation
2
Isolation and Induction of GZMB+ Bregs
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B cells were enriched using Human B Cell Isolation Kit II (Miltenyi Biotec). Separations were performed on an AutoMACS pro Separator following supplier instructions. GZMB+Bregs being a scarce population in HVs with no specific signature, we separated the B cells in two fractions. The first one was left unstimulated in complete medium without stimulation for 72h at 37°C 5%CO2, and the second one was used to induce GZMB expression as previously described (18 (link)): CpG ODN 2006 [1µg/mL] (Invivogen), soluble rhCD40L [50ng/mL] (R&D systems), rhIL-2 [50IU/mL] (Proleukine – Novartis), rhIL-21 [10ng/mL] (R&D systems), anti-human IgG/A/M F (ab)’2 [5µg/mL] (Jackson ImmunoResearch)) in complete medium in 6-well plates at 106 cells/mL for 72h at 37°C 5%CO2. According to this protocol, >95% GZMB+Bregs express GZMB and display suppressive activity (18 (link)). In the following experiments, these cells are thus referred as GZMB+Bregs vs their non-Bregs counterpart.
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