Pe anti mouse ifn γ antibody

Murine bone marrow-derived M0-type macrophages were treated with untreated (control), LPS + IFN-γ (M1), 80 mM NaCl + PBS, 80 mM NaCl + Xiidra, 80 mM NaCl + mIgG, 80 mM NaCl + 7E for 24 h and were collected and stained with FITC anti-F4/80 (Biolegend, Cat# 123108), APC anti-CD86 (Biolegend, Cat# 105012), and PE anti-CD206 (Biolegend, Cat# 141706) antibodies. The expression of F4/80, CD86, and CD206 was analyzed by a CytoFLEX (Beckman Coulter) flow cytometer. Annexin V-FITC Apoptosis Staining/Detection Kit (Abcam, Cat# ab14085) was applied to detect cell death according to the manufacture’s protocol by CytoFLEX (Beckman Coulter) flow cytometer. Cells isolated from conjunctiva or draining lymph nodes were incubated in the RPMI1640 culture medium with PMA (50 ng/ml), ionomycin (1 µg/ml), and Brefeldin A (Golgi Plug) (10 µg/ml). Five hours later, cells were harvested and stained with PE/Cyanine7 anti-mouse CD4 Antibody (Biolegend, Cat# 100422) and fixed by 4% paraformaldehyde. Cells were then treated with permeabilization by BD Cytofix/Cytoperm Plus (BD, Cat# 555028) and stained with PE anti-mouse IFN-γ antibody (Biolegend, Cat# 505807) and PerCP anti-mouse IL-17A antibody (Biolegend, Cat# 506943) and analyzed by CytoFLEX flow cytometer.

Antigen-specific T cell immune responses were assayed on a multicolor flow cytometer (BD Biosciences). After collecting the splenocytes from immunized or unimmunized mice, 2 × 10⁶ cells (100 μL) per sample were stimulated with the SARS-CoV-2 S-protein peptides for 4 h at 37 °C. Brefeldin A (Thermo Scientific) was then added into splenocytes and incubated for 6 h. Stimulated cells were washed in PBS/0.5% BSA and stained with APC/Fire 750 anti-mouse CD3 antibody (BioLegend, San Diego, CA, USA), FITC anti-mouse CD4 antibody (BioLegend), and Brilliant Violet 510 anti-mouse CD8a antibody (BioLegend) surface markers. The cells were then fixed using a Fixation/Permeabilization Solution Kit (BD Biosciences) and stained with PE anti-mouse IFN-γ antibody (BioLegend), PE anti-mouse IL-2 antibody (BioLegend), and PE anti-mouse IL-4 antibody (BioLegend). Data were analyzed with FlowJo software.

The mice were divided into five groups, the PBS group (NS), the OVA (20 µg per mouse) mixed with free CpG (20 µg OVA plus 8 µg CpG per mouse, OVA/CpG) group, the OVA mixed with alum adjuvant (20 µg OVA plus 100 µg Alum adjuvant per mouse, OVA/Alum) group, the OVA (0.2 mg/mL) admixed with the Ncom Gel (20 µg OVA plus 100 µL Ncom Gel per mouse, OVA/Ncom Gel) group and the Ncom Gel only (100 µL Ncom Gel per mouse) group. Female C57BL/6 mice were immunized on the 1^st, 15^th and 22^nd days. Next, spleen single-cell suspensions were obtained from immunized mice seven days after the last immunization, seeded in 24-well plates, restimulated with OVA (50 µg/mL) and incubated for 72 h. After incubation, spleen cells were centrifuged and washed twice, then the cultures were collected for IFN-γ detection. The obtained cells were stained with FITC anti-mouse CD8a antibody and FITC anti-mouse CD4 antibody (Biolegend, San Diego, CA, USA), respectively. Then, the labeled cells were fixed with paraformaldehyde for 30 min and washed twice. Then, the fixed cells were permeabilized with Triton X-100 (Sigma-Aldrich, Saint Louis MO, USA) and stained with PE anti-mouse IFN-γ antibody (Biolegend, San Diego, CA, USA). Finally, the cells were resuspended and analyzed by flow cytometry (ACEA NovoCyte, San Diego, California, USA).