Foxp3 transcription factor permeabilization buffer

IDH-wild type GBM specimens were collected by the Rose Ella Burkhardt Brain Tumor and Neuro-Oncology Center in accordance with the Institutional Review Board (IRB2559) of Cleveland Clinic. Patient demographic information is provided in Table 1. Tumors were cut into small pieces with a razor blade and incubated with collagenase IV (STEMCELL Technologies, Kent, WA) on a rotator at 37°C for 1 hour. Cells were strained over a 40-μm filter and further minced with a plunger to obtain single cell suspensions. Samples were washed with 30 ml PBS twice and treated with RBC Lysis Buffer (Biolegend). Samples were stained with LIVE/DEAD Fixable Stains for 10 minutes on ice and incubated with FcR Blocking Reagent for 15 minutes on ice. Staining with fluorophore-conjugated antibodies was performed in Brilliant Stain Buffer (BD Biosciences) for 20 minutes on ice. Cells were fixed overnight in eBioscience™ Foxp3/Transcription Factor Fixation Buffer. Isotype and Ki-67 staining was performed in eBioscience™ Foxp3/Transcription Factor Permeabilization Buffer with 20 minutes of incubation at room temperature. Samples were acquired with a BD LSR Fortessa. Human MDSC subsets were phenotypically defined based on established guidelines(44 (link)).

Single cell suspensions were washed in PBS prior to resuspension in Zombie Aqua viability dye (BioLegend, San Diego CA) for 15 min at room temperature to exclude dead cells. Primary antibodies (anti-CD11c eFluor610, anti-CD8α Pacific Blue or APC-Cy7, anti-CD11b APC-Cy7, anti-CD103 BV421, anti-MHCII Alexa700 or FITC, anti-CD64 APC, anti-CD45 PE-Cy7, Ly6C BV421, 1A8 PE-Cy7) resuspended in ice-cold FACS buffer (1% bovine serum albumin/0.01% NaN₃ in PBS) were added directly to the cells for 30 min. Pru-RFP parasites were detected in the PE channel, and IL-12p40-YFP was detected in the FITC channel. For intracellular staining, cells were fixed using the FoxP3/transcription factor staining kit fixative (eBioscience, San Diego CA) and subsequently incubated with primary antibodies resuspended in the FoxP3/transcription factor permeabilization buffer (eBioscience). Antibodies used for intracellular cytokine staining include anti-IRF8 PerCP and anti-IRF4 e450 (eBioscience). All samples were run on an LSRII flow cytometer (BD Biosciences, San Jose CA), and the data were analyzed using FlowJo software (FlowJo, Ashland OR).