Macs cd4 cd62l t cell isolation kit

CD62L⁻CD4⁺ T cells (> 95%) from the indicated mouse strains injected with KLH in CFA were purified with MACS CD4⁺CD62L⁺ T-cell isolation kit (Miltenyi) and stimulated with anti-ICOS for the indicated times before fixation, permeabilization and immunostaining. Antibodies or dyes used include: rabbit Bcl-6 (N-3), anti-rabbit Alexa Fluor 568 (for Bcl-6); mouse OPN (AKm2A1, Santa Cruz), anti-mouse Alexa Fluor 647 (for OPN) and nuclear dye DAPI. Images were captured through a 63× objective lens with a Leica SP5X laser scanning confocal microscope and analyzed using ImageJ software, version 1.45b (NIH).

Naïve CD4⁺ T cells (> 95%) were purified from single cell suspensions of B6 spleen using the MACS CD4⁺CD62L⁺ T-cell isolation kit (Miltenyi) and stimulated with anti-CD3 (5 μg/ml) and anti-CD28 (2 μg/ml) for 2 d followed by resting overnight before 20 min of incubation with anti-CD3 (0.2 μg/ml) and/or anti-ICOS (5 μg/ml) and cross-linking with goat anti-hamster Ab (20 μg/ml) for 8 h. RNA was prepared with the RNeasy plus micro kit according to the manufacturer’s instructions (Qiagen). RNA amplification, labeling and hybridization to Mouse Gene 1.0 ST Array (Affymetrix) were performed at the Microarray Core Facility (Dana Farber Cancer Institute), with quadruplicates for anti-CD3 ligation alone and duplicates for anti-CD3 and anti-ICOS co-stimulation. Data were deposited in the GEO database under accession number GSE61284.