Percp cy5.5 conjugated cd45

For human B cell staining, cells were stained with PE-Cy7-conjugated anti-CD19 (BioLegend, #302216), PE-conjugated CD27 (BioLegend, #302808), BV421-conjugated IgD (BioLegend, #348226), BV421-conjugated anti-CD138 (BioLegend, #356516), and APC-conjugated anti-IgG antibodies (BioLegend, #304136). Alternately, cells were permeabilized and stained with primary antibodies against ORAI1, ORAI2, and STIM2 at 37°C for 30 min. Cells were then washed with phosphate-buffered saline (PBS) and incubated with Alexa Fluor^® 488 Goat antirabbit IgG (Thermo Fisher, #A-11008) secondary antibodies at room temperature for 30 min. For mouse cell staining, cells were stained with APC-conjugated B220 (BioLegend, #318326), PE-Cy7-conjugated CD38 (BioLegend, #102718), BV421-conjugated CD138 (BioLegend, #142523), PE-conjugated PD-1 (BioLegend, #135205), APC-conjugated CXCR5 (BioLegend, #145506), PE-conjugated CD95 (BioLegend, #152608), fluorescein isothiocyanate (FITC)-conjugated GL-7 (BioLegend, #144612), APC-Cy7-conjugated-CD4 (BioLegend, #100526), BV510-conjugated-CD3 (BioLegend, #100234), Percp-cy5.5-conjugated-CD45 (BioLegend, #103132), APC-conjugated-CD8 (BioLegend, #100712), PE-Cy7-conjugated-CD19 (BioLegend, #506921) and BV605-conjugated-CD11b (BioLegend, #101257). Samples were analyzed using a BD LSR Fortessa (BD Bioscience).

Flow cytometry was conducted to investigate microglial inflammatory profiles at 3 d post-stroke. Briefly, single-cell suspensions were obtained from contralateral (CL) and ipsilateral (IL) brain tissues using an enzymatic tissue dissociation kit (Miltenyi Biotech Inc., Gaithersburg, MD, USA) before centrifuging through a 30/70 Percoll (GE Healthcare, Chicago, IL, USA) gradient solution to remove myelin, as described before [13 (link),26 (link)]. Cells were subsequently stained with BUV395-conjugated CD11b (1:400, BD Biosciences, San Jose, CA, USA), PerCP-Cy5.5-conjugated CD45 (1:400, BioLegend, San Diego, CA, USA), eFluor450-conjugated CD16/32 (1:400, Affymetrix eBioscience, San Diego, CA, USA), FITC-conjugated CD206 (1:400, BioLegend, San Diego, CA, USA), Alexa 700-conjugated CD86 (1:400, BD Biosciences, San Jose, CA, USA), PE-conjugated Ym1 (1:400, Abcam, Boston, MA, USA), and BV605-conjugated CD68 (1:400, BioLegend, San Diego, CA, USA) antibodies for 20 min at 4 °C in the dark. At least 10,000 events were recorded from each hemispheric sample using an LSR Fortessa flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with FlowJo software v10.4. Please see Supplementary Materials for detailed methods.