Foxp3 transcription factor staining buffer set

Myeloid and lymphoid subsets were isolated from the tumors and quantitatively analyzed as previously described (Chowdhury et al., 2019 (link)) with some modifications. Briefly, after mechanical homogenization, the tumors were digested with collagenase A (1 mg ml−1; Roche) and DNase I (0.5 μg ml−1; Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% L-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) for 45 minutes shaking (150 rpm) at 37 °C. Cells were filtered through 100 μm cell strainers, washed in isolation buffer and stained. Myeloid cells were stained immediately, and lymphoid subset underwent a separation gradient using Percoll (67%, 40%), followed by staining. Dead cells were excluded by staining with Ghost Dye cell viability reagent. Extracellular antibodies included: anti-B220 (BD) (1:200), anti-CD19 (Tonbo) (1:200), anti-CD45 (BD and Biolegend) (1:400), anti-CD4 (BD) (1:400), anti-CD8 (Tonbo) (1:400), anti-NK1.1 (BD) (1:300), anti-CD11b (BD) (1:500), anti-CD11c (BD) (1:200), anti-F4/80 (Tonbo) (1:500), and anti-MHC class II (Tonbo) (1:400) antibodies. Intracellular antibodies included: anti-CD3e (BD) (1:400), anti-TCRβ (BD) (1:300) and anti-FOXP3 (Thermo) (1:300). Cells were fixed using the FOXP3/transcription factor staining buffer set (Tonbo) according to the manufacturer’s protocol. Samples were analyzed using a BD LSRFortessa cell analyzer.