Ic fixation and permeabilization buffer

For cytokine detection, in vitro restimulation was performed for 20 h with 20 µg/ml of T. gondii lysate antigen in supplemented Iscove’s DMEM at 37°C with 5% CO₂. Protein transport inhibitor (Thermo Fisher Scientific) and labeled anti-CD107a were added for the last 4 h of incubation. Following surface staining, cells were fixed and permeabilized using IC Fixation and Permeabilization buffer (Thermo Fisher Scientific) according to manufacturer’s instructions.
IL-21 staining was carried out using an rIL-21R/Fc fusion protein (R&D Systems) followed by PE-conjugated F(ab’)2 goat anti-human Fc (Jackson ImmunoResearch Laboratories) according to a previously published protocol (19 (link)).

Cells were stained with fluorochrome-labeled anti-mouse Ab such as CD45, CD3, CD4, CD8, Foxp3, IFN-γ, IL-17A, CD11b, CD11c, pAKT, pSTAT3, CCR6, or MHCII. For intracellular cytokine staining, single-cell suspensions from the tumor and TDLNs were stimulated using a cell stimulation cocktail (eBioscience, San Diego, California, USA, 500X used at 1X) consisting of PMA (40.5 μM, Cayman, Ann Arbor, Michigan, USA), ionomycin (670 μM, BioVision, San Francisco, USA), and protein transport inhibitors-brefeldin A (5.3 mM, Thermo, Massachusetts, America) and monensin (1 mM, Thermo, Massachusetts, America) for 6 h at 37 °C and 5% CO₂. After 6 h, the cells were harvested and washed, surface stained with CD45, CD3, CD4, CD8, CD11b, CD11c, CCR6, and MHCII (FACS Buffer, Thermo, Massachusetts, America), fixed, permeabilized (IC fixation and Permeabilization buffer, Thermo, Massachusetts, America), and stained for pAKT, pSTAT3, IFN-γ, and IL-17A (Thermo, Massachusetts, America). Isotype controls with the same fluorochrome were used as controls. Cells were acquired using the FACS Aria II machine and analyzed using FlowJo software.