Lymphoprep separation medium

CD4⁺ T cells were isolated from human PBMC or buffy coats after centrifugation using Lymphoprep separation medium (Corning, Vienna, VA) and a negative selection kit according to the manufacturer’s protocol (either #130–091-155 from Miltenyi Biotech (Bergisch Gladbach, Germany) or #17952 from Stem Cell Technologies (Vancouver, Canada)). Purity of isolated cells was typically >95%, and cells were resuspended in complete RPMI medium (10% FCS, 2mM glutamine (#25030–149, Gibco), 50U/ml Penicillin + 50μg/ml streptomycin (#15070–063, Gibco)). Purified CD4⁺ T cells were activated for the indicated time points in 96- or 48 well culture plates (Greiner, Monroe, NC) precoated with antibodies to CD3 (clone OKT3) and CD46 (clone TRA2.10)^{64 (link)} at 1.5–2 ×10⁵ cells/well (96well) or 3.5–5×10⁵/well (48 well) in media containing 50 U/ml recombinant human IL-2 (Peprotech) in an incubator at 37°C and 5% CO₂. Cells were harvested and assayed at the indicated timepoints.
For restimulation experiments, cells were harvested after 36–48 hours of activation and rested for 5 days in 5U/ml recombinant human IL-2 (Peprotech), then harvested, recounted and re-stimulated in plates precoated with CD3 and CD46 antibodies in media containing 50 U/ml recombinant human IL-2 (Peprotech) for 18 hrs.

Human monocytes were isolated from PBMCs (obtained from freshly
drawn blood after centrifugation using Lymphoprep separation medium
(Corning) to generate a buffy coat) using the MACS human CD14⁺Positive Monocyte Isolation Kit (130–050-201, Miltenyi Biotech),
according to the manufacturers’ instructions. Purity of isolated
monocyte populations was typically > 96 %. Monocytes were then
cultured in 24-well plates for 6hr to over-night at a concentration of 5.0
× 10⁵ cells/well/ml media with or without the addition of
100 ng/ml) LPS. For measurement of impact of LFA-1 activation, monocytes
were cultured in 24-wells that had been coated overnight with recombinant
ICAM-1-Fc (2.0 μg/ml of each in PBS).