Pe cyanine7 anti f4 80

After the kidney tissue was dissociated by Liberase TM (Roche) in RPMI-1640 medium at 37°C for 20 minutes, 100 μL of the single-cell suspensions was incubated with the corresponding antibodies at room temperature for 30 minutes in the dark. The following antibodies were purchased from BioLegend: FITC anti-CD45 (catalog 103108), APC/Cyanine7 anti-CD11b (catalog 101226), PE/Cyanine7 anti-F4/80 (catalog 123114), and APC anti-Ly6c2 (catalog 128016). PE anti-Arg-1 (catalog IC5868P) was purchased from R&D Systems. The data were analyzed by using Kaluza.

Cells were suspended in 1 ml of FACS buffer (PBS with 2% FBS). After Fc receptor blocking with antimouse CD16/32 (BioLegend), cells were incubated with a mix of antibodies specific to analysis at 1:200 dilution each for 20 min, and dead cell marker 7-aminoactinomycin D (5 µl/sample) or DAPI (1 µl/sample) was added right before acquisition on a BD FACSCanto II device. Data were analyzed using BD FACSDiva software. Cell counts were reported to initial cell number determined after Percoll density gradient purification.
The antibody panel for CNS-infiltrating cells was as follows: Alexa Fluor 488 anti-CD45, APC anti-CD3, PE anti-CD11b, PE–cyanine 7 anti-F4/80, and 7- aminoactinomycin D (BioLegend). The antibody panel for RNAseq was as follows: Alexa Fluor 488 anti-CD45, APC anti-CD3, PE anti-CD11b, and DAPI (BioLegend).