Anti cd80 v450

Splenic lymphocytes were stained for surface markers as follows. For extracellular markers, single cells were stained at 2 × 10⁶ cells per well in a 96-well V bottomed plate. T cells were stained with anti-CD3 Alexa 700 (BD, clone 500A2), anti-CD4 PerCP (BD, clone RM4-5), anti-CD44 FITC (BD, clone IM7), anti-CD62L V450 (BD, clone MEL-14), and anti-FOXP3 APC (BD, clone MF23). B cells were stained with anti-CD19 PEcy7 (eBiosciences, clone 6D5), anti-B220 FITC (eBiosciences, clone RA3-6B2), anti-CD21 APC (BD, clone 7G6), anti-CD23 PE (BD), and anti-CD80 V450 (BD, clone 16-10A1). Fifty microliters of the antibody master mix prepared in MACS buffer (1× PBS, 2 mM EDTA, and 0.5% BSA) was added per well in all staining procedures. Cells were acquired on an LSRII (Becton Dickinson) and analyzed by FlowJo (Tree Star, Ashland).

For direct immunofluorescence labeling, macrophages were stained with the following fluorescence-labeled anti-human mAbs: anti-CD40-FITC, anti-CD64-A700, anti-CD80-V450 and anti-CD86-APC, anti-CD11b-pacific blue, anti-CD163-PE, anti-CD206-FITC (all from BD Biosciences, San Diego, CA) and anti-CD200R-APC Abs (Serotec, Oxford, UK). For Notch1 receptor analysis, macrophages were immunostained using rabbit anti-Notch1 (1:100 dilution, ab52627 from Abcam, Cambridge, UK) Abs and Alexa-488 labeled anti-rabbit IgG as secondary antibodies. For apoptosis analysis, cells were incubated with Annexin V/propidium iodide following the manufacturer’s recommendations (Life Technologies). Cells stained with, isotype-matched, irrelevant Abs were used as negative controls. Fluorescence was measured by flow cytometry after gating on FSC/SSC parameters using a FACS LSR II® (BD Biosciences) and next analyzed using FlowJo® VX software (Tree Star, Inc., Ashland, OR, USA).