Intracellular staining perm wash buffer

For flow cytometry analysis, adherent cells were recovered by trypsinising or gentle scraping and washed in PBS with 2 mM EDTA (PBS/EDTA). Non‐adherent cells were recovered from culture supernatants by centrifugation for 5 min at 300 g and washed once in PBS/EDTA. Cells were stained with fixable Zombie UV Live/Dead dye (BioLegend) for 6 min at room temperature. Excess stain was quenched with FBS‐complemented DMEM. For MDMs, Fc‐blocking was performed with PBS/EDTA+10% human serum for 10 min at 4°C. Cell surface with CD86‐Bv711 (IT2.2, BioLegend) and HLA‐DR‐PerCpCy5.5 or PE‐Cy7 (L243, BioLegend) staining was performed in PBS/EDTA at 4°C for 30min. Unbound antibody was washed off thoroughly, and cells were fixed in 4% PFA prior to intracellular staining. For intracellular detection of SARS‐CoV‐2 nucleoprotein, cells were permeabilised for 15 min with Intracellular Staining Perm Wash Buffer (BioLegend). Cells were then incubated with 1 μg/ml CR3009 SARS‐CoV‐2 cross‐reactive antibody (a kind gift from Dr. Laura McCoy) in permeabilisation buffer for 30 min at room temperature, washed once and incubated with secondary Alexa Fluor 488‐Donkey‐anti‐Human IgG (Jackson Labs). All samples were acquired on a BD Fortessa X20 or LSR II using BD FACSDiva software. Data were analysed using FlowJo v10 (Tree Star).

After the incubation period, cells were washed with 2 mL buffer [phosphate-buffered saline (Sigma) containing 5% fetal bovine serum (Life technologies)] and centrifuged at 350 × g for 5 min. Cell surface staining of monocytes was done in 0.1 mL buffer for 30 min on ice with fluorescein isothiocyanate-conjugated antibody to human CD14 (clone HCD14, BioLegend) at a dilution of 1:20. Cells were then washed and fixed in 0.5 mL/tube Fixation Buffer (BioLegend) in the dark for 20 min at room temperature before centrifugation at 350 × g for 5 min and then washed again. The fixed cells were re-suspended in 0.1 mL Intracellular Staining Perm Wash Buffer (BioLegend) and stained with 10 μL phycoerythrin-conjugated antibody to human IL-23p19 (clone #727753; R&D Systems) in the dark for 20 min at room temperature. Finally, another washing step was done before the fixed and stained cells were re-suspended in 0.3 mL buffer. Cells were analyzed on a BD FACS Accuri C6 flow cytometer. Individual populations were gated according to forward scatter (FSC), side scatter (SSC), and specific markers, and the data were subsequently analyzed with FlowJo X software.

Apoptosis was detected by staining cells with Annexin V-FITC/propidium iodide (Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. CD14Ab (Biolegend, CA, USA) staining was used to exclude Mφs from the analysis.
Expression of active caspase-3 was measured using a Fluorescein Active Caspase-3 Staining Kit (Invitrogen, Carlsbad, CA, USA). Expression of CD206, CD163, and CD86 was measured by direct immunofluorescence using PE-conjugated CD206 (Biolegend, CA, USA) and CD163 (Biolegend, CA, USA) and APC-conjugated CD86 (Biolegend, CA, USA). An isotype control was used to exclude nonspecific signals.
For intracellular CCL2 staining, cells were stimulated with Leukocyte Activation Cocktail (BD Bioscience, CA, USA) for 4 h at 37 °C. The cells were then fixed and permeabilized with Fixation Buffer and Intracellular Staining Perm Wash Buffer (Biolegend, CA, USA) according to the manufacturer’s instructions. Next, the cells were stained with a PE-mouse anti-human MCP-1 antibody (BD Pharmingen, San Diego, CA, USA). Data were acquired with a FACScan flow cytometer (BD Biosciences, San Diego, CA, USA) and analyzed using FlowJo 7.6.1.

The detailed flow cytometry procedure was performed as previously described [31 (link)]. The following antibodies were used: anti-mouse IFNγ (BD Biosciences), anti-mouse CD45 (BD Biosciences), anti-mouse Gr-1 (TONBO Biosciences), anti-mouse Ly6G (TONBO Biosciences), anti-mouse ly6C (BD Biosciences), anti-mouse CD11b (TONBO Biosciences), anti-mouse F4/80 (BioLegend), anti-human CD11b (TONBO Biosciences), anti-human CD33 (BD Biosciences), and anti-human HLA-DR (BD Biosciences). Detailed information on the antibodies is provided in Additional file 2: Table S2. Briefly, the cells were digested and suspended as single cells, washed with PBS, and then resuspended in cell stabilizing buffer (BioLegend cat. No. 420201); the supernatant was discarded, and the sample was centrifuged at 350 g for 5 min. Then, 5 μl of Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301) and the antibodies were added and cultured for 15-20 min at room temperature. Finally, the cells were assessed by flow cytometry. For intracellular staining of IFNγ, fixation buffer (BioLegend Cat. No. 420801) was added and incubated for 20 min at room temperature. Then, the cells were washed with Intracellular Staining Perm Wash Buffer (BioLegend Cat. No. 421002), resuspended and centrifuged. IFNγ antibody was added, and the cells were cultured for 20 min and finally assessed by flow cytometry.

Brain tissues were placed into 500 ug/ml collagenase type IV (Solarbio, China; dissolved in RPMI 1640 medium containing 10% FBS) and cut into several pieces with a surgical scalpel. After digestion at 37°C for 1 h, cells were pressed through a 70‐μm cell strainer. Myelin debris was removed, and single‐cell suspension was acquired by gradient density centrifugation (30%/70% Percoll solution; GE Healthcare, USA). For surface staining, the brain cells were incubated with CD45‐APC‐Cy7 (Biolegend, USA) and CD4‐FITC (Biolegend, USA) for 30 min. For intracellular staining, cells were fixed and permeabilized with Fixation Buffer (Biolegend, USA) and Intracellular Staining Perm Wash Buffer (Biolegend, USA) according to the manufacturer's guidelines, followed by GZMB‐APC/Per‐Cy7 (Biolegend, USA) staining for 40 min. The eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (00‐5523‐00; Thermo Scientific) and Foxp3‐PE antibody (Biolegend, USA) were utilized for intranuclear staining in Treg cells. Flow cytometry analysis was performed by CytoFLEX cytometer (Beckman, CA). The data were analyzed with CytExpert and FlowJo software.