Fitc anti mouse cd3ε

Mouse splenocytes were cultured in 12-well round bottom plates (4×10⁶ cells/well) and stimulated with or without mixed T. gondii peptides (4 μg/ml/peptide) for 24 h. Cells were treated with BD GolgiStop™ protein transport inhibitor (BD Biosciences, San Diego, USA) for 6 h post stimulation. Then, cells were recovered and blocked with anti-mouse CD16/32 (Biolegend, CA, USA). After washing twice with cell staining buffer (BD Biosciences), cells were prepared in 50μl staining buffer and stained with FITC anti-mouse CD3ε (Biolegend, CA, USA) and with APC anti-mouse CD8α (Biolegend, CA, USA) at 4°C for 30 min. Following two additional washes, cells were thoroughly suspended in 250 μl fixation/permeabilization solution (BD Biosciences) for 20 min at 4°C. Then cells were washed, re-suspended in 50μl of BD Perm/Wash solution and stained with PerCP/Cy5.5 anti-mouse IFN-γ (Biolegend, CA, USA) at 4°C for 30 min. Finally, IFN-γ expression in CD8⁺CD3⁺ T cells was measured by BD Accuri™ C6 flow cytometer.

To quantify the counts of eosinophils, cells from BALF or lung tissues were resuspended in 100 µL RPMI 1640 medium containing BV510 Live/Dead (catalog no. 423101, Biolegend), Alexa Fluor®-700 anti-mouse CD45 (catalog no. 103127, clone 30-F11, Biolegend), KIRAVIA Blue 520^TM anti-mouse F4/80 (catalog no. 123161, clone BM8, Biolegend), Brilliant Violet 421^TM anti-mouse CD11c (catalog no. 117329, clone N418, Biolegend), APC/Cyanine7-anti-mouse CD11b (catalog no. 101226, clone M1/70, Biolegend), and PE anti-mouse CD170 (Siglec-F) (catalog no. 155505, clone S17007L, Biolegend) on ice for 30 minutes. To quantify the counts of T lymphocytes, cells from BALF or lung tissues were resuspended in 100 µL RPMI 1640 medium containing BV510 Live/Dead (catalog no. 423101, Biolegend), FITC anti-mouse CD3ε (catalog no.100306, clone 145-2C11, Biolegend) on ice for 30 min. All cells were washed with PBS and were measured on a BD LSRFortessa^TM Flow Cytometer (BD Biosciences). All antibodies were purchased from Biolegend and were diluted at 1:200. A minimum of 5000 events per plot were collected and analyzed using FlowJo V10 software. The numbers presented in the flow cytometry analysis images are percentage-based. All antibodies were validated for the specified application by respective manufacturer.