Brilliant violet 421 anti mouse human cd11b antibody

Blood was anticoagulated by 15 g/L EDTA-Na₂ and then centrifuged at 1500 rpm for 5 min to remove plasma. Cell populations were resuspended in precooling PBS and then stained using APC anti-mouse Ly-6 g antibody (BioLegend, Cat. No. 127614, dilution 1:100, San Diego, USA) and Brilliant Violet 421 anti-mouse/human CD11b antibody (BioLegend, Cat. No. 121035, dilution 1:100, San Diego, USA) for 30 min at 4 °C. Red blood cells were removed with Red Blood Cell Lysis Buffer (Solarbio, Cat. No. R1010, Beijing, China). Flow cytometry was performed on the Beckman Coulter platform (CA, USA). The neutrophil proportion was analyzed using Kaluza software (Version 2.1).

Isolated brain mononuclear cells from the mock- or ZIKV-infected mice in cell staining buffer (PBS with 1% FBS and 0.09% NaN₃) were stained for 30 min with fluorescence-conjugated antibodies, namely, Brilliant Violet 421 anti-mouse/human CD11b antibody (101236; BioLegend, San Diego, CA), PE/Cyanine7 anti-mouse CD45 Antibody (103114; BioLegend), APC anti-mouse TNF-α antibody (506307; BioLegend), FITC anti-mouse IL-6 monoclonal antibody (MP5-20F3; 11-7061-82; eBioscience, San Diego, CA), PE anti-mouse IL-1β antibody (12-7114-82, eBioscience), and Alexa Fluor 488 anti-flavivirus group antigen antibody (4G2; NBP2-52709AF488; Novus Biologicals, Englewood, CO). The cells were then analyzed using a FACSAria III sorter (BD Biosciences, San Jose, CA), and data were analyzed using FlowJo software (BD Biosciences). All the fluorochromes were compensated for.

Isolated brain mononuclear cells from the mock or SARS-CoV-2-infected mice in cell staining buffer (phosphate-buffered saline [PBS] with 1% FBS and 0.09% NaN₃) were stained for 30 min with fluorescence-conjugated antibodies, namely, brilliant violet 421 anti-mouse/human CD11b antibody (101236, BioLegend, San Diego, CA, USA), PE/Cyanine7 anti-mouse CD45 antibody (103114, BioLegend), APC anti-mouse TNF-α antibody (506307, BioLegend), FITC anti-mouse IL-6 monoclonal antibody (MP5-20F3) (11-7061-82, eBioscience, San Diego, CA, USA), FITC anti-human ACE2 antibody (NBP2-7211F, Novus Biologicals, Centennial, CO, USA), and Alexa 647 anti-Iba1 antibody (78060S, Cell Signaling Technology, Danvers, MA, USA). Cells were then analyzed by FACSAria III sorter (BD Biosciences, San Jose, CA, USA), and data were analyzed by FlowJo software (BD Biosciences). All fluorochromes were compensated. The total leukocyte population was gated for microglia (CD11b⁺, CD45^low). For annexin V staining, mock or SARS-CoV-2-infected HMC3 were stained with FITC-recombinant human annexin V, following the protocols of Annexin V-FITC Apoptosis Detection kit (BMS500FI-20, eBioscience).