Foxp3 fix perm solution

PBMCs (1 × 10⁶ cells/ml) resuspended in 50 µl of PBS were stained with anti-human CD3 PE cy7, anti-human CD4 Violet and anti-human CD25 APC (clone: PC61; Biolegend, UK) for 30 min at 4 °C. Post incubation, the cells were washed in PBS twice and fixed with Foxp3 Fix Perm solution (Thermo Fischer) for 30 min at room temperature, followed by a wash and staining with anti-human Foxp3 PE (clone: PCH101; Thermo Fischer) in diluted permeabilization buffer (Thermo Fischer) for 30 min at 4 °C. Post incubation, the cells were washed and analysed using a Cyan ™ ADP flow cytometer (Dako). Regulatory T cells were defined as CD3⁺ CD4⁺ CD25⁺ Foxp3⁺ cells [ gating strategy Supplementary Fig. 1] [32 (link)].

Frozen PBMCs were thawed at 37 °C and washed in 10 mL of RPMI containing FCS (10%) (Sigma Aldrich). The pelleted cells were re-suspended in PBS (1 × 10⁶ cells/mL), were stained with combinations of antibodies (Supplementary Table S1) for 30 min at 4 °C and followed by 2 washes with PBS. For intracellular transcription factor staining for regulatory and follicular helper T cell staining, cells were surface-stained (anti-human CD3, anti-human CD4) and fixed with Foxp3 Fix Perm solution (eBiosciences, San Diego, CA, USA) for 30 min. This was followed by washing the cells, permeabilization with diluted permeabilization buffer (eBiosciences) and staining with antibodies for anti-human foxp3 and anti-human bcl6 for 30 min at 4 °C followed by 2 washes with PBS.
Samples were acquired using a Cyan ADP flow cytometer (Dako, Glostrup, Denmark). Data analysis was done using Summit V 4.3 software. Spectral overlap when using more than one colour was corrected via compensation. Appropriate isotype controls were used for setting gates. The gating strategy used to identify the T cell subset has been shown in Figure S1; the gating strategy for B cell subset distribution has been published [35 (link),36 (link)]. The detailed methods for stimulation of PBMCs to induce cytokine production by CD4 T cells and staining for toxin expressing immune cells can be found in the Supplementary Methods.

Frozen PBMCs were thawed using complete RPMI (RPMI+10%FBS) and washed with PBS. The cells were then treated with cisplatin for 5 mins, followed by incubation with metal-conjugated surface antibodies cocktail (Table 1) for 30 mins at 37°C. Cells were washed twice with CyFACS buffer (PBS with 4% FBS, 0.05% sodium azide), followed by primary antibody staining for 30 mins on ice. Subsequently, cells were washed twice with CyFACS buffer, followed by permeabilization and fixation with Foxp3 fix/perm solution (eBioscience) for 30 mins on ice. Following this, cells were washed with permeabilization buffer (Biolegend) and then stained with intracellular antibody cocktail (Table 1) for 30 mins on ice, wash with Biolegend permeabilization buffer then stained with metal-conjugated streptavidin for 10 mins on ice. Finally, cells were washed with PBS and fixed overnight using 2% PFA made in PBS. The next day, cells were barcoded and stained with Cell-ID Intercalator-Ir (Fluidigm) in PBS for 20 mins at room temperature. Cells were washed twice with CyFACS buffer followed by a final wash using MiliQ water and passed through size filter. Filtered cells were analyzed using Helios mass cytometer (Fluidigm) with CyTOF software version 7.0.8493. Data analyses were performed using Flowjo V10.5.3 (BD) and Cytofkit2 (33 (link)).