Intracellular staining perm wash buffer 10x

Cell surface expression levels of FcεRI, c-kit, TLR4, and ST2 were determined by flow cytometric analyses using FITC-labeled anti-mouse FcεRIα, APC-labeled anti-mouse CD117, PE-labeled anti-mouse CD284 (TLR4) (clone SA15-21, BioLegend), and FITC-labeled anti-mouse T1/ST2 (clone DJ8, BioLegend), respectively. For intracellular staining, a fixation buffer (#420801, BioLegend) and an intracellular staining perm wash buffer (10X) (#421002, BioLegend) were used according to the manufacturer’s instructions with PE-labeled anti-mouse FcεRIα. Brefeldin A (#420601) and Monensin (#420701) were purchased from BioLegend. Data collected using a MACSQuant Analyzer (Miltenyi Biotec, Bergisch Glandbach, Germany) or a FACSLyric (Becton Dickinson, Franklin Lakes, NJ, USA) were evaluated with FlowJo software (Tomy Digital Biology, Tokyo, Japan).

Tumors collected from mice were divided into small pieces and homogenized in cold staining buffer to form single-cell suspensions in the presence of digestive enzyme. Cells were stained with fluorescence-labeled antibodies against CD45 (Biolegend, Cat. no. 103116, clone 30-F11), CD3 (Biolegend, Cat. no. 100217, clone 17A2), CD4 (Biolegend, Cat. no. 100429, clone GK1.5), CD8a (Biolegend, Cat. no. 100721, clone 53–6.7), Foxp3 (Biolegend, cat. no. 126404, clone MF-14), NK-1.1 (Biolegend, Cat. no. 108709, clone PK136), CD62 L (Biolegend, Cat. no. 104407, clone MEL-14), CD44 (Biolegend, Cat. no. 103005, clone IM7), CD11b (Biolegend, Cat. no. 101205, clone M1/70), CD206 (Biolegend, Cat. no. 141731,clone C068C2), CD80 (Biolegend, Cat. no. 104733, clone 16-10A1), F4/80 (Biolegend, Cat. no. 123131, clone BM8), Ly-6G (Biolegend, Cat. no. 127615, clone 1A8), Gr-1 (Biolegend, Cat. no. 108407, clone RB6-8C5), CD86 (Biolegend, Cat. no. 105011, clone GL-1), CD103 (Biolegend, Cat. no. 121415, clone 2E7), and CD11c (Biolegend, Cat. no. 117307, clone N418), and were treated with Fixable Viability Dye (Biolegend, cat. no. 423101), Cell Stimulation Cocktail (Biolegend, cat. no. 423303), Intracellular Staining Perm Wash Buffer (10X) (Biolegend, Cat. no. 421002), and Fixation Buffer (Biolegend, Cat. no. 420801).