Purified Hamster Anti-Mouse CD80

Protocol tips

Upstream tips	Protocol tips	Downstream tips
DCs were generated from mouse BM using an established protocol (31) with minor modifications (32). Briefly, mouse BM was obtained from the long bones of the hind legs. After erythrocyte lysis, BM cells were suspended at 1×106/ml in RPMI 1640 with 10% FBS, 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4. Cells were plated into wells of six-well tissue culture plates, and cultured for 6 days	Ab incubation for 30 min at 4°C

Upstream tips
DCs were generated from mouse BM using an established protocol (31) with minor modifications (32). Briefly, mouse BM was obtained from the long bones of the hind legs. After erythrocyte lysis, BM cells were suspended at 1×106/ml in RPMI 1640 with 10% FBS, 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4. Cells were plated into wells of six-well tissue culture plates, and cultured for 6 days
Protocol tips
Ab incubation for 30 min at 4°C

Publication protocol

DCs were generated from mouse BM using an established protocol (31) with minor modifications (32). Briefly, mouse BM was obtained from the long bones of the hind legs. After erythrocyte lysis, BM cells were suspended at 1×106/ml in RPMI 1640 with 10% FBS, 10 ng/ml murine GM-CSF and 1 ng/ml murine IL-4. Cells were plated into wells of six-well tissue culture plates, and cultured for 6 days; the purity of CD11c+ DCs was about 85%. Day 6 DCs were stimulated for 24–48 hours with Alternaria extract (25–50 µg/ml), or poly I:C (25 µg/ml) or combinations thereof. In separate experiments, we further purified DCs with a magnetic cell separation system (MACS; Miltenyi Biotech, Auburn, CA) and anti-CD11c (N418) immunomagnetic beads (Miltenyi Biotec). These highly purified DCs (>97% purity) behaved similarly to those without MACS purification. Because these extra manipulations might have affected DC functions, we used BM-derived DCs without MACS purification in this study. The functional responses of DCs to Alternaria extract and poly I:C were assessed by expression of cell surface molecules by FACS, quantitative real-time PCR and Western blotting. Cytokine production was analyzed by ELISA. For FACS analysis, after stimulation for 24 h (MHC Class II, CD40, CD80, CD86) or 48 h (OX40L), DCs were preincubated with Fc-receptor blockers (anti-CD16/CD32) for 30 min at 4°C and stained with PE-conjugated anti-CD11c (clone HL3) and FITC-conjugated anti-MHC Class II I-Ad (AMS-32.1), anti-CD40 (HM40-3), anti-CD80 (16-10A1), and anti-CD86 (GL1), or biotinylated anti-OX40L (RM134L) plus FITC-conjugated streptavidin for 30 min at 4°C. FITC-conjugated mouse IgG2b, Armenian hamster IgG2, rat IgG2a and rat IgG2a, and biotinylated Armenian hamster IgM were used as isotype controls. All Abs used for DC FACS analysis were obtained from BD Pharmingen. After washing, DCs were resuspended in PBS containing 1% BSA and 0.1% NaN3, fixed with 1% paraformaldehyde, and analyzed by a FACScan flow cytometer (BD Immunocytometry Systems, Mountain View, CA) by gating on a CD11c-positive forward scatter-high population. For cytokine analysis, cell-free supernatants were collected after 24 h stimulation; concentrations of IL-6, IP-10, I-TAC and IFN-β were measured by ELISA kits (R&D Systems) according to the manufacturer’s directions.

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