Foxp3 tf staining buffer set

Anti–CD3ε-PerCP (100326), anti–CD8a-AF700 (100730), anti–CD8a-APC (100712), anti–PD-1-PE/DZ594 (109116), anti–Ki67-FITC (652410), anti–TCF1-PE (655208), anti–IFN-γ-BV421 (505830), anti–IFN-γ-PerCp/Cy5.5 (505822), anti–TNF-α-APC/Cy7 (506344), anti–TIM-3-PE/Cy7 (119716), anti–CD45-BV510 (103138), anti–Ly108 (Slamf6)-PE (134606), anti–PD-1-FITC (135214), anti–CD45.1-PE (110708), anti–CD45.2-AF488 (109816), purified anti-mouse CD3ε (100340), and purified anti-mouse CD28 (102116) were obtained from Biolegend. Anti–CD8-FITC (D271-4) and tetramer-SIINFEKL-APC (TS-5001-2c) were purchased from MBL. Cell Stimulation Cocktail Plus Protein Transport Inhibitors and FOXP3/TF Staining Buffer Set were bought from eBioscience. Naive CD8a⁺ T Cell Isolation and Tumor Dissociation Kits were obtained from Miltenyi Biotec.

The yolk sac was removed from embryos by gentle aspiration in 0.5× Ginzburg Fish Ringer solution (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO₃, without Ca²⁺). Embryos were digested in CO₂-independent media (Life Technologies, Cat# 18045088) containing 3 mg/mL of collagenase II (Santa Cruz, Cat#sc-506177) at 37 °C under gentle rotation. The cell suspension was passed through a 70 μm cell strainer (BD, Cat#352350), washed with PBS and pelleted at 2800×g. Cells were stained for viability using ZombieRed Fixable Viability Kit (Biolegend, Cat# 423110) for 20 min at RT, protected from light. Cells were washed with FACS buffer (PBST, 1% BSA w/v), fixed and permeabilized with the FoxP3/TF Staining Buffer Set (eBioscience, Cat#00-5523-00) according to the manufacturer’s instructions. Cells were stained with 1 μg/mL Hoechst 33258 (Thermo Fisher Scientific, Cat# 33258) at 4 °C for 15 min and analysed using the BD Fortessa II and FACSDiva Software on a linear scale. Wild-type fish treated with 0.1 μg/mL nocodazole or 3 μM etoposide were used as G2/M and S phase inhibitor controls, respectively.

A single-cell suspension of tonsil mononuclear cells was stained for surface markers in 100 µl PBS/BSA for 15 min at 37 °C. After washing, biotin was stained with fluorochrome-labeled streptavidin for 10 min at 4 °C. Afterward, cells were fixed for 35 min at RT, permeabilized, and TF were stained at RT for 45 min using the Foxp3/TF staining buffer set (eBioscience). Measurements were performed on a FACSymphony (BD Bioscience) and data analyzed with FlowJo v.10.6.01 software (TreeStar). The antibody panel used for flow cytometry is shown in Supplementary Table 3. Flow cytometry was performed according to Guidelines for the use of flow cytometry and cell sorting in immunological studies⁵⁰ .

OT1 T cells were incubated with 50 μl of Live/Dead Fixable aqua dead (Thermo Fisher; 1:300 dilution) for 30 min in PBS at 25 °C, washed and then incubated again with 50 μl of FCR blocking reagent (clone 2.4G2, BD Biosciences; 1:50 dilution) for 30 min at 4 °C. Cells were washed and incubated at 4 °C for another 30 min with surface markers directed with PE/Cy5.5 anti-mouse CD3e (clone 2. 145-2C11; 1:100 dilution), BV711 anti-mouse CD8α (clone 2. 53-6.7, BioLegend; 1:50 dilution) and PE/Dazlee anti-mouse CD45.1 (clone A20, BioLegend; 1:100 dilution). For intracellular staining, the following antibodies were used: PE anti-human hIgG4-Fc (clone HP6025, Abcam; 1:50 dilution) for detecting the PD-1 IgG4 decoy and PE anti-mouse IL-33 (clone 396118, Invitrogen; 1:25 dilution). After surface staining, genetically engineered OT1 cells were washed twice and fixed/permeabilized using the Foxp3 TF staining buffer set (Thermo Fisher Scientific; 1:4 dilution). Cells were washed and resuspended in PBS supplemented with 2% BSA and 0.01% azide (FACS buffer) to be acquired with a BD flow cytometer LSR II cytometer with FACSDiva software v8.0.1 (BD) and analyzed using FlowJo software v10.7. Because Pd1d was a common module, we used it to evaluate the transduction efficiency across conditions. Next, we added UT OT1 cells to get the same number of transduced cells in all the groups.