Rat anti mouse cd16 cd32 mab 2.4g2

Draining lymph nodes were suspended in sterile phosphate-buffered saline and then pooled to have enough tissues and cells. Cell suspensions of DLNs from adults and from neonates were first pre-treated with purified rat anti-mouse CD16/CD32 mAb (2.4G2 BD Pharmingen Cat No. 553141) to block FcγRs II/III and then stained for 30 min on ice with the following fluorochrome-conjugated Abs: rat anti-mouse CD19-APC (1D3 BD Pharmingen Cat No. 550992), rat anti-mouse CD138-PE (281-2 BD Pharmingen Cat No. 553714), and rat anti-mouse Gr-1-PercP (RB6-8C5 BD Pharmingen Cat No. 552093). Cells were then washed, re-suspended in Cytofix/Cytoperm Kit (BD Pharmingen Cat No. 54714) on ice for 10 min, and washed with 1× Perm Wash Buffer 1:10 (BD™ Phosflow Cat No. 557885). Cells were then intracellular stained with goat anti-mouse IgG-FITC (Jackson Labs Cat No. 115-095-205). Cells were acquired on a CyAn™ ADP Analyzer. Data were analyzed with FlowJo software v7.6.5 (TreeStar).

BALF was obtained as previously described [29 (link)]. Briefly, 1 mL of saline with 100 µM EDTA was intratracheally injected and aspirated by a 24-G catheter with a 1 mL syringe. This procedure was repeated 4 times and recovered BALF was pooled.
After Fc blocking with 20 µg/mL of rat anti-mouse CD16/CD32 mAb (2.4G2, BD, Franklin Lakes, NJ, USA), BALF cells were stained with PE-conjugated rat anti-mouse CD68 mAb (FA-11, BioLegend, San Diego, CA, USA), BV421-conjugated rat anti mouse CD86 mAb (GL-1, BioLegend), APC-conjugated rat anti mouse CD206 mAb (C068C2, BioLegend), BV510-conjugated rat anti-mouse CD3 mAb (17A2, BioLegend), FITC-conjugated rat anti-mouse CD19 mAb (1D3/CD19, BioLegend), APC/Cy7-conjugated rat anti-mouse CD45 mAb (30-F11, BioLegend), and PE/Cy7-conjugated rat anti-mouse CX3CR1 mAb (SA011F11, BioLegend). A flow cytometric analysis was performed using BD LSRFortessaTM (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo software ver. 10.7.1 (BD Biosciences).