Foxp3 fix perm buffer kit

Total PBMCs were stimulated with α-CD3 antibody (OKT3, Biolegend) at 0.1 μg/ml for 2 days in complete RPMI 1640 media containing 2% heat-inactivated human AB serum (Sigma-Aldrich), washed, and rested in complete media for 5 days. On day 7, cells were harvested and 200 k cells were cultured in complete media containing only IL-2 mutein at titrating concentrations in a 96-well flat bottom plate. Five days later, cells were harvested and labeled with a viability dye (LIVE/DEAD Fixable Near-IR Dead Cell stain Kit, ThermoFisher Scientific) per manufacturer's protocol and stained for cell surface markers: CD3 (BUV805, BD Biosciences), CD4 (BUV395, BD Biosciences), CD8 (BV510, Biolegend), CD25 (BB515, BD Biosciences), and CTLA4 (a-CD152-PE/Cy7, Biolegend). Stained cells were fixed and permeabilized using the Foxp3 fix/perm buffer kit (eBioscience) and stained for Foxp3 (Alexa Fluor 647, Biolegend) and Ki67 (BV421, BD Biosciences). Samples were acquired on BD FACSymphony (BD Biosciences) and analyzed using FlowJo software.
The raw values of the percent Ki67-positive or Foxp3 and CTLA4 median fluorescence intensity were converted to percent response (% response) based on the values obtained from cells stimulated with wildtype IL-2 at 66.7 nM or media only (baseline). The equation that was used for conversion is as follows:

Mice were challenged, and tumor-infiltrating lymphocytes (TILs) from tumors were isolated as described above. TILs were stained with anti-CD45.2, anti-CD11b, and anti-F4/80 antibodies to purify CD11b⁺ F4/80⁺ TAMs by flow sorting. 5–7 × 10⁴ WT conventional naïve T cells were incubated with TAMs isolated from different treatment groups. These cells were stimulated in vitro in a 96-well round bottom plate using anti-CD3 (1.25 µg/ml) and anti-CD28 (1.25 µg/ml) antibodies for 48 h. Monensin (0.5 μl/ml) and brefeldin A (0.5 μl/ml) (BD Biosciences) were added during the final 4 h of stimulation. After stimulation, the cells were stained with surface markers, fixed, and permeabilized using the FoxP3 Fix/Perm buffer kit (eBioscience) as per the manufacturer’s instructions. The cells were then stained with antibodies for intracellular proteins for further analysis using flow cytometry.

Apoptosis was quantified using cellevent caspase 3/7 green detection reagent (ThermoFisher). Cells were incubated with 3 μM of the reagent for 30 min at 37°C. To separate out nTreg, mTreg, and emTreg, cells were blocked with PBS 2% FBS + human IgG 10 μg/ml and stained with the surface antibodies CD45RA-pacific blue, CD95-BV510 (BioLegend), and HLA-DR-APC-Cy7 (BD) for 30 min at room temperature. Then, cells were fixed and permeabilized with FOXP3 perm/fix buffer kit (ThermoFisher) for 1 h at 4°C, followed by intracellular staining with anti-Ki67-PerCP-Cy5.5 (BD), anti-ICOS-BV605, and anti-CTLA4-BV711 (BioLegend). Samples were acquired on a Fortessa flow cytometer (BD). Flow cytometry data were analyzed using the Flowjo software, version 10.6.2 (BD). For some experiments, bulk Treg cells were cultured in X-VIVO media in the presence of caspase 3/7 green reagent and anti-CD45RO (Alexa Fluor 700; BioLegend) and followed for 24 h by time-lapse imaging, using Nikon AXR Inverted Confocal Laser Scanning Microscope (Nikon Corporation, Minato City, Tokyo, Japan).

PBMC or CD4+ T cells from PWH were thawed and stained with live dead blue (Thermofisher) for 15 min at RT. Cells were washed with PBS 2% FBS, resuspended in blocking buffer (PBS 2% FBS 20 µg/ml human IgG) and stained with the surface antibodies: CD45RA, CD95, CD25, CD39, HLA-DR, CD69, CD3, CD4, and CD127 (see Table S1). 10 µl of brilliant stain buffer (BD) was added to each sample. Cells were incubated for 30 min at RT and washed with PBS 2% FBS. Cells were fixed and permeablized with FOXP3 perm/fix buffer kit (Thermofisher) for 1h at 4°C, followed by intracellular staining with FOXP3, ICOS, Ki67, CTLA-4, TNF-α and IFN-γ (see Table S1) for 30 min at 4°C. Samples were acquired on the Aurora flow cytometer (Cytek, Bioscience, CA). PBMC from two healthy controls (HC) were included in every run to check for possible batch effect. Flow cytometry data were analyzed using the Flowjo software v10.6.2.