Fitc mouse anti mouse 1 e k mhc class 2 antibody clone 14 4 4s

SRDC and PECs were stimulated as described above (2.5.). Supernatant was centrifuged at 300×g to pool floating cells and attached cells after their detachment using accutase (Affymetrix eBioscience). After centrifugation at 300×g, supernatant was removed for quantification of cytokines (section 2.5.) and pelleted SRDC were suspended and saturated for 30 min on ice in PBS containing 1% bovine serum albumin, 2% mouse serum and 0.1% azide (PBS-BSA-azide). After centrifugation, 3 × 10⁵ SRDC were incubated for 30 min on ice in the dark in PBS-BSA-azide with 0.5 μg fluorescein isothiocyanate (FITC) mouse anti-mouse H–2K[k] MHC class I antibody (clone 36-7-5, BD Pharmingen™), 0.5 μg FITC mouse anti-mouse I-E[k] MHC class II antibody (clone 14-4-4S, BD Pharmingen™), or 0.5 μg FITC mouse IgG2a, κ isotype control (clone G155-178, BD Pharmingen™). After centrifugation, SRDC were suspended in 300 μL PBS-2% paraformaldehyde and analysed by flow cytometry (BD FACSCalibur™ and CellQuest™ software, BD Bioscience). PECs were incubated for 15 min at 4 °C in the dark in 100 μL binding buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 5 mM CaCl₂) with 5 μL annexin V-FITC (BD Pharmingen™) and 5 μg/mL propidium iodide (Sigma). After centrifugation, PECs were suspended in 300 μL binding buffer and analysed by flow cytometry.