Cd45 buv395 clone 30 f11

Immune cells were isolated at time of euthanasia from P2 and P3 mouse blood by Ammonium-Chloride-Potassium (ACK) lysis of red blood cells at room temperature (63 (link)). Cells were washed with RPMI 1640 (Gibco), pelleted at 300g for 5 min at 4°C, and resuspended in magnetic-activated cell sorting (MACS) buffer [1× PBS (pH 7.2) + 2 mM EDTA + 0.5% BSA]. Cell suspensions were first stained with eBioscience Fixable Viability Dye eFluor 506 (Thermo Fisher Scientific/Invitrogen, catalog no. 65-0866-18) in PBS for 30 min at room temperature. Cells were stained with the following antimouse surface antibodies in MACS buffer for 30 min at room temperature: F4/80-BV785 (clone BM8; BioLegend, catalog no. 123141); CD45-BUV395 (clone 30-F11; BD Biosciences, catalog no. 564279); CD11b-FITC (clone M1/70; Thermo Fisher Scientific/Invitrogen, catalog no. 11-0112-41) or CD11b-PE (clone M1/70; BD Biosciences, catalog no. 557397); and Ly6G-APC (clone 1A8-Ly6g; Thermo Fisher Scientific/Invitrogen, catalog no. 17-9668-82). After surface antibody staining, the cells were fixed (30 min at room temperature) using the FoxP3 fixation/permeabilization kit (Thermo Fisher Scientific, catalog no. 00-5523-00). Stained cells were analyzed on a BD LSRFortessa (BD Biosciences) using the BD FacsDiva software (v9). Data were analyzed with BD FlowJo software version 10.9.

Single cell suspensions from naïve or day 14 hr. polygyrus-infected C57BL/6 mice were stained with CD45-BUV395 (clone 30-F11, BD Biosciences), CD64-Al647 (clone X54-5/7.1, Biolegend), MHCII-PE (clone M5/114.15.2, BD Biosciences), CD19-BB515 (clone 1D3, BD Biosciences), CD3-BV605 (clone 17A2, Biolegend), CD4-Pac Blue (clone RM4-5, BD Biosciences) and DAPI. 700,000 CD45+ cells were sorted into FACS buffer using a BD Influx cell sorter (BD Biosciences) followed by purification of B cells, CD4 T cells and macrophages. 5,000 cells of each population were sorted into 100 µl RNA lysis buffer (Zymo Research) and stored at −80C. RNA was extracted using the Quick-RNA MicroPrep Kit (Zymo Research) and its quality assessed using the High Sensitivity RNA Screen Tape (Agilent) and a 4150 TapeStation System (Agilent) according to the manufacturer’s instructions.