Pe cyanine7 anti mouse cd4 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.

Flow cytometry was performed using the following fluorochrome-conjugated antibodies: CD3e Monoclonal Antibody (145-2C11) (Thermo Fisher Scientific, Waltham, USA), PE/Cyanine7 anti-mouse CD4 Antibody (BioLegend, San Diego, USA), anti-mouse CD8a Antibody (BioLegend, San Diego, USA), CD25 Monoclonal Antibody (PC61.5) (Thermo Fisher Scientific, Waltham, USA), PE/Cyanine7 anti-mouse TCR β chain Antibody (BioLegend, San Diego, USA), Purified anti-mouse/human IL-5 Antibody (BioLegend, San Diego, USA), Ki-67 Monoclonal Antibody (SolA15)(Thermo Fisher Scientific, Waltham, USA), and FOXP3 Monoclonal Antibody (FJK-16s) (Thermo Fisher Scientific, Waltham, USA). A Foxp3 Transcription Factor Staining Set was purchased from eBioscience (Thermo Fisher Scientific, Inc.). A Zombie Aqua Fixable Viability Kit was obtained from BioLegend. Collagenase-IV, 17β-estradiol (E2), DNase I, sesame oil, phorbol 12-myristate 13-acetate (PMA), and ionomycin were purchased from Sigma-Aldrich (St. Louis, MO, USA).