Foxp3 transcription factor fixation permeabilization concentrate and diluent

Unless otherwise noted, tissue plasticware was purchased from Corning (In Vitro Technologies, Auckland, New Zealand); all cell culture reagents were from Gibco BRL (Thermo Fisher Scientific, Auckland, New Zealand). Alexa Fluor 488 anti-H2AX-Phosphorylated (Ser139) Antibody was from BioLegend (Norrie Biotech, Auckland, New Zealand). Rabbit anti-8-OHdG polyclonal antibody (J-1: sc-139586) was from, Santa Cruz Biotech (Dallas, Texas, USA) and isotype control (IgG/10500C) was from Thermofisher Scientific (Wellington, New Zealand). Goat Polyclonal Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody was from Abcam (Cambridge, MA, USA). Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent was purchased from eBioscience (Huntingtree Bioscience Supplies, Auckland, New Zealand). Click-iT EdU Alexa Fluor Flow Cytometry Assay Kits, Vybrant DyeCycle Stains, MitoSOX™ Red Mitochondrial Superoxide Indicator and MitoTracker^® Red CMXRos were purchased from Life Technologies (Thermo Fisher Scientific, Auckland, New Zealand). CellTiter 96^® AQueous One Solution Cell Proliferation Assay (MTS) was sourced from Promega Corporation (Madison, WI, USA). Luminescent ATP Detection Assay Kit was obtained from Abcam (Cambridge, MA, USA). Sodium ascorbate, and all other chemicals and reagents were from Sigma Chemical Company (St. Louis, MO, USA).

C26 tumors were mechanically and enzymatically homogenized using buffer containing RPMI with 1 mg/mL collagenase (Sigma C0130) and 0.1 mg/mL DNase (Sigma D4527). Cells were stained for flow cytometry according to reagent manufacturers’ protocols. Viability was determined using eBioscience e780 fixable viability dye at a 1/1,000 dilution. All antibodies are listed in the Supplemental Experimental Procedures. CD49b positivity was determined using a fluorescence-minus-one control. Anti-CD16/32 (unconjugated; 2.4G2; 5 μg/mL; BD) was used for Fc blocking prior to antibody staining. The eBioscience Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent was used prior to Foxp3 staining. Sample analysis was performed using an LSR II cytometer. At least 200,000 events per tumor sample were collected. Data were subsequently analyzed using FlowJo.

For surface staining, cells were labeled with mAbs against various targets including CD8, CD62L, CCR7, CD45RA, CD45RO, CD25, CD44, OX40, PD-1, CTLA-4, and KLRG-1 (Ebiosciences), incubated at 4 °C for 15 minutes, then washed twice with PBS 1% FBS (FACS buffer), and finally fixed in PBS containing 1% paraformaldehyde (Fix buffer). For T-bet (4B10) and Eomes (Dan11mag), and Foxp1 intracellular staining, cells were first labeled with surface markers CD8, CD62L, OX40 (Ebiosciences) for T-bet and Eomes, or CD25 (BD Biosciences), CD8, CD27, and CD44 (Ebiosciences) for Foxp1 detection, respectively, and then fixed and permeabilized with Foxp3/Transcription Factor Fixation/Permeabilization Concentrate and Diluent (Ebiosciences) in a 96-round-well plate. Intracellular labeling for T-bet and Eomes (Ebiosciences), and Foxp1 (LifeSpan Technologies) was performed according to manufacturer’s instruction. For evaluation of Perforin, Granzyme B, and IFN-ɤ cytokine secretion, effector cells were incubated with brefeldin A for 4 hours at 37 °C to disrupt Golgi-mediated transport and accumulate cytokines. Cells were then surface stained, permeabilized with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (BD Biosciences), and intracellularly stained as recommended. Flow cytometry data was analyzed using FlowJo Version 10.0.7 software.

The protocol for cytokine production and intracellular accumulation for flow cytometric detection was adopted from C. L. Fellman et al. and modified.[46 (link)] Cells were treated with 25 ng/ml Phorbol-12-Myristate-13-Acetate (PMA, Sigma-Aldrich) and 500 ng/ml ionomycin (Sigma-Aldrich). After 3 hours of incubation, 1 μg/ml Brefeldin-A (Sigma-Aldrich) was added and cells were cultured for additional 3 hours. Cells were then washed, stained with a viability dye (Fixable Viability Dye eFlour^®780, eBioscience), fixed and permeabilized (Foxp3/Transcription factor fixation/permeabilization concentrate and diluent, eBioscience) and stained with the following primary conjugated antibodies: anti-canine CD3-AlexaFluor488 (clone CA17.2A12, Leukocyte antigen biology lab, UCD), anti-canine CD4-PE (CA13.1E4, Leukocyte antigen biology lab, UCD) and anti-human IL17-AlexaFluor647 (Goat anti-human IL17, R&D systems). Anti-IL17 antibody was conjugated using Alexa Fluor 647 monoclonal antibody labeling kit (Invitrogen, Carlsbad, CA) per manufacturer’s instructions. Fluorescence was detected by a flow cytometer (Cytomics FC500, Beckman Coulter) and flow cytometry data were analyzed using FlowJo flow cytometry software (Tree Star Inc.).