Cd4 macs beads

APC-labeled I-E^k tetramers in complex with MCC, or CLIP were obtained from the NIH facility. All washes and incubations were done in modified RPMI-1640 containing 2% FCS and 0.075% sodium bicarbonate. For tetramer staining, peripheral CD4⁺ T cells were enriched by CD4⁺ MACS beads (Miltenyi Biotech, Germany), and CD4SP thymocytes were enriched by negative selection of CD8⁺ MACS beads (Miltenyi Biotech). Enriched CD4⁺ peripheral T cells or CD4SP thymocytes were stained with APC-conjugated MCC tetramer or PE-conjugated Hb tetramer on ice for about 1.5–2 hr. The APC-conjugated or PE-conjugated CLIP tetramers were used as a negative control. In the case of MCC tetramer staining, the cells were also stained for APC/Cy7-conjugated CD4, PE/Cy7-conjugated CD8, and Pacific blue-conjugated CD3 antibodies. Cells positive for LIVE/DEAD Fixable Blue Dead Cell Stain were gated out of the analysis.

Human CD4⁺ cells were isolated from peripheral blood from healthy volunteers using Ficoll® Plaque Premium and positive selection on CD4⁺ MACS beads (Miltenyi) as previously described (Martinelli et al., 2008 (link)). CD4⁺T cells were resuspended in RPMI-1640 supplemented with 10% FCS, 100 U/ml penicillin, 100 ​μg/ml streptomycin,1 ​mM sodium pyruvate, 1 ​mM nonessential amino acids, 2 ​mM l-Glu, and 50 ​μm β-mercaptoethanol supplemented with 25 ​ng/mL of IL-2 at a density of 500,000 ​cells/mL.