Anti mouse cd16 cd32 mab 2.4g2

Spleen cells were incubated with anti-mouse CD16/CD32 mAb (2.4G2; BD Biosciences, San Jose, CA) in FACS buffer (HBSS containing 2% FCS) for FcR blocking for 15 min on ice, and then incubated with BV421-conjugated anti-mouse CD3 mAb (145-2C11; Biolegend, San Diego, CA), APC-conjugated anti-mouse CD8 mAb (53-6.7; Biolegend), PE-conjugated anti-mouse CD4 (GK1.5 Biolegend) or Siglec F (E50-2440; BD Biosciences) mAb, PE-Cy7-conjugated anti-mouse CD19 mAb (6D5; Biolegend) or Gr1 (RB6-8C5; Affymetrix eBioscience, San Diego, CA) mAb for 20 min on ice. The expression profile of each cell surface marker on 7-aminoactinomycin D (7-AAD; Sigma-Aldrich Co. LLC, St. Louis, MO) -negative cells was analyzed on a MACSQuant Analyser (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSQuantify Software (Miltenyi Biotec) and FlowJo software (Tree Star Inc., Ashland, OR).

Isolated liver mononuclear cells (LMNCs) were re-suspended at a concentration of approximately 1 × 10⁶ cells per 50 μL in FACS staining buffer containing anti-mouse CD16/CD32 mAb (2.4G2) from BD Pharmingen (San Jose, CA, USA) to block nonspecific binding to Fcγ receptors and incubated for 10 min at 4°C. Cells were stained with live/dead Fixable Blue Dead Cell Stain Kit from Life Technologies (Grand Island, NY, USA) to gate out dead cells. Further, we added the antibodies: CD11b APC, NK1.1 PE, CD3 Pacific Blue, Ly6C FITC (eBioscience, San Diego, CA, USA) to the cells and incubated for 30 min in the dark at 4°C. For the negative control, the cells were stained with isotype-matched control antibodies (eBioscience, San Diego, CA, USA). The cells were washed with FACS staining buffer by centrifugation at 1,500 rpm for 5 min at 4°C and fixed with 1% paraformaldehyde. The cells were acquired on a BD LSR II instrument (BD Biosciences, San Jose, CA, USA) and the data was analyzed by FlowJo software.

Skin DC migration was determined as described elsewhere^{28 (link)}. In brief, mice were epicutaneously treated with 40 µl of 0.5% (w/v) FITC isomer I (Sigma-Aldrich Co. LLC) in a 1:1 mixture of acetone (Wako Pure Chemical Industries, Ltd., Osaka, Japan) and dibutyl phthalate (Sigma-Aldrich Co. LLC) (20 µl to each surface of the left ear) and the vehicle alone (20 µl to each surface of the right ear). Twenty-four hours later, submaxillary lymph nodes (LNs) were separately collected from both the FITC-treated left and vehicle-treated right ears. After incubation with anti-mouse CD16/CD32 mAb (2.4G2, BD Biosciences) for 15 minutes on ice, LN cells were stained with PE-conjugated anti-mouse CD11c mAb (HL3, BD Biosciences) and APC-conjugated anti-mouse MHC Class II (I-A/I-E) mAb (M5/114.15.2, Affymetrix eBioscience) for 20 minutes on ice. The percentage of FITC-positive cells among 7-AAD-negative MHC Class II^hi CD11c⁺ cells was determined using a FACS Verse (BD Biosciences), and data analyses were performed using FlowJo software (Tree Star Inc.).