Foxp3 alexa fluor 700

To study the immune cells in distant tumors, right tumors were harvested from mice in different groups (n = 5) and stained with Viobility 405/520 Fixable Dye (Miltenyi), CD45.2 APC-CY7 (Biolegend, Clone: 104), CD3e FITC (Biolegend, Clone: 17A2), CD8a PE-vio615 (Miltenyi, Clone: REA601), PD-1 PE (Biolegend, Clone: 29F.1A12), TIM3 APC (Miltenyi, Clone: REA602), CD4 VioBlue (Miltenyi, Clone: REA604), and Foxp3 Alexa Fluor 700(Biolegend, Clone: MF-14.1A12) antibodies, according to the manufacturer's protocols.
Briefly, tumor tissues were cut into small pieces and digested with collagenase and DNase. Then, cell suspension was filtered through a 75-μm cell mesh and resuspended in PBS (pH 7.4) with 0.5% FBS for further analysis. Flow cytometric analysis was performed using a FACS LSRFortessa flow cytometer (BD).
Tumor-infiltrating cytotoxic T lymphocytes (CTL) and helper T cells were CD45⁺CD3⁺CD4⁻CD8⁺ and CD45⁺CD3⁺CD4⁺CD8⁻, respectively. Then, the expressions of PD-1 and TIM-3 in cytotoxic T lymphocytes were analyzed. Further, CD4⁺ helper T cells were classified into regulatory T cells (Tregs) (CD3⁺CD4⁺Foxp3⁺) and effective T cells (CD3⁺CD4⁺Foxp3⁻).

Therapeutic anti-CTLA-4 (clone 9H10) antibody and isotype control antibody was purchased from BioXcell (cat: BE0131 and BE0087). Antibodies used for flow cytometry were purchased from the following sources (dilutions are indicated in parentheses): eBioscience (CD45.2 Alexa Fluor 700, cat: 56-0454 (1:200), CD3 PE-Cy7, cat: 25-0031 (1:200), CD4 ef450, cat: 48-0041 (1:200), CD4 APC-efluor780, cat: 47-0041 (1:400), CD8 PerCP-efluor710, cat: 46-0083 (1:200), CD11b APC-efluor 780, cat: 47-0112 (1:600), ICOS PE, cat: 12-5985 (1:200), ICOSL PE, cat: 12-5985 (1:200), CTLA-4 PE, cat: 12-1522 (1:200), NK1.1 PE, cat: 12-5941 (1:200), IFNγ PE, cat: 12-7311 (1:200), FoxP3 Alexa Fluor 700, cat: 56-5773 (1:100), FoxP3 APC, cat: 17-5773 (1:200), GATA-3 PE, cat: 12-9966 (1:100), RORγT PerCP-efluor710, cat: 46-6981 (1:100), Tbet PE-Cy7, cat: 25-5825 (1:100), EOMES efluor 450, cat: 48-4875 (1:100), PD-1 PE-Cy7, cat: 25-9985, (1:200)), Biolegend (CD3 BV570, cat: 100225 (1:100), CD11b BV570, cat: 101233 (1:50), Bcl-6 Alexa 594, cat: 648308 (1:50)), Invitrogen (Granzyme B PE-Texas Red, cat: GRB17 (1:125), Granzyme B APC, cat: GRB05 (1:125)) and BD Pharmingen (Ki-67-Alexa Fluor 488, cat: 561165 (1:50), CXCR5-biotin, cat: 551960 (1:100)).