Cd4 and cd8 isolation kits

Splenocytes were isolated from mice at day 8 following i.p. infection with 2.5 × 10⁵ PFU of the DM strain of mouse hepatitis virus (MHV-DM). Enriched populations of CD4+ and CD8+ T cells, isolated according to the manufacturer’s instructions (CD4 and CD8 Isolation kits, Miltenyi Biotec, Auburn, CA, USA), were labeled with the fluorescent dye, carboxyfluorescein diacetate succinimidyl ester (CFSE) (Life Technologies, Grand Island, NY, USA), at 2.5 μM final concentration. Then 1 × 10⁶ total cells per well were incubated with FTY720P 100 nM or vehicle and stimulated with 5 μM final peptide concentration of CD4+ T cell immunodominant epitope M133-147, CD8+ T cell immunodominant epitope S510-518, or non-specific OVA control, and cultured for 72 h at 37°C, 5% CO₂ in complete media. Cells were then washed and the Fc receptor blocked with 1 × PBS containing 1% BSA and a 1:200 dilution of rat anti-mouse CD16/32 antibody (Pharmingen, San Jose, CA, USA). Next, cells were stained for surface antigens using APC-conjugated rat anti-mouse CD4 and CD8 (Pharmingen, San Jose, CA, USA), according to the viral peptide stimulation condition, for 45 min at 4°C. Cells were analyzed and the data assessed as described above.

CD4+ and CD8+ T cells were isolated from fresh apheresis material (Sanquin) of healthy volunteers upon informed consent using the ELUTRA™ cell separation system (Gambro) and CD4+ and CD8+ isolation kits (Miltenyi Biotec). moDCs were plated in 96-well plates (25,000 DCs; ratio 1:8) and rested at 37°C, 5% CO₂ for 2 h. moDCs were stimulated overnight with 50 µg/ml LPS in the absence or presence of 10 nM C5a, after which 200,000 CD4+ or CD8+ T cells were added. Co-cultures were incubated at 37°C, 5% CO₂, and supernatants were collected after 6 days of stimulation.