Anti cd16 32 antibody clone 2.4g2

Cells were blocked with anti-CD16/32 antibody (clone 2.4G2, Tonbo Biosciences) and then stained using antibodies against αM (M1/70 PE), αV (RMV-7 PE), β2 (M18/2 FITC), and β3 (2C9.G2 APC) integrins from Biolegend. PE-conjugated antibodies against α4, α5, and β1 integrins were obtained from Santa Cruz Biotechnology. Isotype controls were purchased from the corresponding vendor, and Fc block antibody was from Tonbo. For blocking, LEAF-grade antibodies against β1 (HMβ1-1), β2 (M18/2), and β3 (2C9.G2) integrins and matching isotype controls were purchased from Biolegend. Thorough washing was performed to remove excess, unbound antibodies. Flow cytometry was performed on a BD LSRII flow cytometer using BD FACSDiva software (BD Biosciences). Post-processing was performed in FlowJo (Treestar), and further data analysis and quantification was performed in R. Cell populations were gated on forward and side scatter to select for intact, single cells. Acquisition was performed until at least 10 000 events were collected using a preliminary analysis gate or until the sample was exhausted. Statistical analysis was performed considering p < 0.05 to be statistically significant. Data were analyzed using a one-way ANOVA followed by Tukey's HSD post-hoc test.

Lung mononuclear cells were obtained as previously described ^{2 (link)}. Cells were stimulated for 6 hours with Phorbol-12-myristate 13-acetate (PMA) (50 ng/ml), ionomycin (0.5 μM), and monensin (2 μM), and pre-incubated with anti-CD16/32 antibody (clone 2.4G2; TONBO Bioscience, Kobe, Japan). For intracellular staining, cells were fixed with Transcription Factor Buffer Set (BD Bioscience, San Jose, CA, USA) in accordance with the manufacturer’s protocol.

Colonic lamina propria cells were incubated with anti-CD16/32 antibody (clone 2.4G2, Tonbo Biosciences, San Diego, CA, USA) to block Fc receptors and then stained with fluorescein isothiocyanate-conjugated anti-CD4 antibody (clone RM4-5, Biolegend, San Diego, CA, USA). Subsequently, the cells were fixed and permeabilized with true-nuclear transcriptional factor buffer set (Biolegend) then stained with phycoerythrin (PE)-conjugated anti-Foxp3 antibody (clone MF-14, Biolegend), PE-conjugated anti-RORγt antibody (clone B2D, eBioscience, San Diego, CA, USA) or Alexa fluor 647-conjugated anti-T-bet antibody (clone 4B10, Biolegend). After washing the cells with phosphate-buffered saline containing 0.5% bovine serum albumin, the fluorescence intensity was measured by flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA). Data were analyzed using CellQuest software (BD Biosciences).