Anti cd3 coated 96 well plates

We purchased anti-CD3-coated 96-well plates from BD Biosciences. Roswell Park Memorial Institute (RPMI)-1640 culture medium was obtained from GibcoBRL Corp. Rabbit anti-human CD4 and CD8 were purchased from Dako. Biotinylated anti-human CD25 was obtained from Sigma. The following antibodies and QDs were purchased from Invitrogen: biotinylated anti-mouse IgG, anti-rabbit IgG (H + L)-conjugated QD 655, and streptavidin-conjugated QD 605. Additionally, before the antibody labeling, all QDs were centrifuged to remove aggregates of QDs.

CD4+ T cells were isolated from PBMCs by the Dynabeads FlowComp Human CD4 kit (Invitrogen). Purity was consistently >95% of live cells as determined by CD3 and CD4 staining by flow cytometry. CD4+ T cells were grown in 96-well plates or anti-CD3 coated 96-well plates (BD) with or without 20 μM minocycline hydrochloride (Sigma) in 100 μl RPMI 1640 supplemented with 10% FBS, 2 mM L-glutamine, 1 mM HEPES buffer, and 2 mg/mL gentamicin. After 24 hours, a 50 μL aliquot containing 60 μM minocycline was added to replenish minocycline at a final concentration of 20 μM in the 150 μL well volume. Some wells were also stimulated with 1,000 U/mL each of IFNα-2a and IFNβ-1a (PBL). All wells had a final volume of 150 μL and were cultured for an additional 24 hours (total 48 hours) before flow cytometry analysis.