Cd3 apc clone sk7

PBMC/T-cells were stimulated with 1 μg/mL purified plate-bound anti-CD3 mAb (clone Hit-3a; BioLegend) and soluble 0.3 μg/mL anti-CD28 mAb (clone CD28.2; eBioscence) for 18 hours in complete RPMI medium in 96 flat-bottom wells with a starting concentration of 2 × 10⁵ cells/well (1 × 10⁶/mL) of PBMCs or 1 × 10⁵ cells/well (1 × 10⁶/mL) of T-cells at 37° C, 5% CO₂. For neutrophil/T-cell suppression assay, neutrophils were cocultured with CD3⁺ PBMC/T-cells at a 3 : 1 ratio at 37° C, 5% CO₂.
CD25 surface expression was assessed using flow cytometry. The following mAbs were used for PBMC/T-cell staining: CD4-PE-Cy7 (clone SFCI12T4D11), CD8-APC A750 (clone B9.11) (Beckman Coulter), CD3-APC (clone SK7) (BD Biosciences), and CD25-PerCP-Cy5.5 (clone BC96) (BioLegend).
To detect IFN-γ by intracellular staining, brefeldin (10 μg/mL) was added for the last 4 hours of incubation. The cells were washed, fixed, permeabilized using Intracellular Fixation and Permeabilization Buffer Set (eBioscience) according to the manufacturer´s protocol, and stained with IFN-γ-BV421 (clone 4S.B3) (BioLegend).

Primary human NK cells were isolated from peripheral blood mononuclear cells (PBMC) obtained by Ficoll-Paque separation from buffy coats provided by the Finnish Red Cross. NK cells were isolated using a human NK cell isolation kit (Miltenyi Biotec). NK cells from three donors were pooled to achieve enough material for DSRT and RNA sequencing. The purity of NK cells was evaluated by flow cytometry using CD56-PE (clone NCAM16.2) and CD3-APC (clone SK7) staining (BD Biosciences), and the final pool contained >90% CD56⁺CD3⁻ cells in all replicates. Primary bone marrow mononuclear cells (BM MNC) were isolated from bone marrow aspirates from healthy donors using Ficoll-Paque separation. All normal NK cells were cultured in R10 supplemented with 2.5 ng/mL recombinant human IL-2 (Peprotech). BM MNC were cultured in Mononuclear Cell Medium (PromoCell). To obtain actively proliferating normal NK cells, NK cells were expanded using an artificial antigen-presenting cell K562 variant (K562-aAPC) as previously described^{39 (link)}. Briefly, PBMC were isolated from buffy coats by Ficoll-Paque separation and co-cultured with irradiated (100 cGy) K562-aAPCs at a ratio of 1:2 (PBMC:aAPC) in RPMI1640 with 50 IU/mL IL-2 at 200,000 PBMC/mL. Cultures were refreshed with half-volume media changes every two to three days, and re-stimulated with aAPCs at ratio of 1:1 every seven days.