Foxp3 transcription factor staining kit

Cells were stained with the indicated antibody cocktail (Supp. Tables 1 an 2) for 30 min in PBS (Gibco) with 10% FBS (Sigma) at 4°C. Following staining, cells were pelleted at 500g for 10 min and washed 3 times in 2ml PBS. Cells were fixed for 20 min (Foxp3 Transcription factor Staining kit, Tonbo). Samples were additionally stained for 30 min for intracellular markers before analysis. Samples were run on a 5-laser Becton Dickinson LSR Fortessa X-20. Analysis was carried out in FloJo V10 by Treestar. Samples with <60% viability were excluded from analysis (mean 78.8%).

Three days after Th17 cells differentiation, cells were restimulated with 50 ng/ml PMA, 1 μM ionomycin, and 0.07% of Golgi Stop for 4 h. Following restimulation, cells were stained with viability dye (Ghost Dye UV 450; Tonbo Biosciences), blocked with an anti-FcR Ab (clone 2.4G2), and surface stained with APC-Cy7 anti-CD3 (clone 145-2C11), FITC anti-CD4 (clone GK1.5), BV786 anti-CD90.2 (clone 53-2.1), and APC anti-IL-23R (clone 12B2B64). Cells were then fixed, permeabilized using the Foxp3/transcription factor staining kit (Tonbo Biosciences), and intracellularly stained PE-Cy7 anti–IL-17A (clone eBio17B7). Flow cytometry analysis was conducted on LSR II flow cytometer, and all data were processed using FlowJo software version 10. Antibodies were purchased from Thermo fisher (Invitrogen). In select experiments, Cell Tracer Violet dye was added per manufacturer's instructions (ThermoFisher) prior to Th17 cell differentiation. Th17 cell proliferation was determined by flow cytometry 3 days later by gating on viable, CD4+ IL-17A+ cells and measuring the proliferation index using FlowJo software.

Tumors and lungs were minced and dissociated in RPMI-1640 media (Corning #MT10040CV) containing 2.5% FBS, 1 mg/ml collagenase IA (Sigma-Aldrich #C9891), and 0.25 mg/ml DNase I (Sigma-Aldrich #DN25) for 45 minutes at 37°C. Digested tissue was then filtered through a 70-µm strainer, and red blood cells were lysed using ACK Lysis Buffer (KD Medical #RGF-3015). Samples were washed with PBS and stained with Ghost Dye Violet V510 (Tonbo Biosciences #13-0870) to exclude dead cells. After washing with buffer (0.5% BSA, 2mM EDTA in PBS), samples were blocked in αCD16/32 mouse Fc block (Tonbo Biosciences #70-0161) and stained for extracellular proteins using an antibody master mix made in buffer. After washing with buffer, cells were fixed with 2% PFA. For FoxP3 intracellular staining, cells were permeabilized using the FoxP3 Transcription Factor Staining Kit (Tonbo Biosciences #TNB-0607-KIT) per manufacturer protocol. Flow cytometry data was obtained on a BD 4-laser Fortessa using BD FACS Diva software v8.0.1 and analyzed using FlowJo software v10.6.1. Fluorescence minus one (FMO) samples were used as gating controls when needed. Antibodies used in flow panels are detailed in
Table 1, and gating strategies used in analysis are detailed in
Table 2. Each data point is generated after analyzing at least 5×10
⁵ viable cells from a specimen from an individual mouse.