GSM8172823: Sample_18_MGPhag_Raggc_antiIL17; Mus musculus; RNA-Seq
Mice were euthanized in a CO2 chamber and transcardially perfused with ice-cold Hanks' Balanced Salt Solution (HBSS, Thermo Fisher, 14175103). Spleens were mashed with the plunger of a syringe and treated with 1ml of ACK for 10min buffer to remove erythrocytes. Splenic cells were washed, pelleted and stained with the following rat antibodies: BV421 anti-CD45 (1:100, Biolegend, 103133), PE-Cy7 anti-mouse CD11b (1:300, eBioscience, 50-154-54), PerCP/Cy5.5 anti-mouse Ly-6C (1:300, Biolegend, 128012), Alexa Fluor 488 anti-CD115 (1:100, Biolegend, 135512) and Alexa Fluor 700 anti-Ly6g (1:100, Biolegend, 127621) in PBS with 2% FBS. After washing, CD115–CD11b+Ly6cintLy6g+ and CD115–CD11b+Ly6cintLy6g+/Tdt neutrophils were sorted using BD FACSAriaTM II (BD Bioscience). For analysis of Foxp3 and intracellular staining, splenic neutrophils were treated with an eBioscience FoxP3-staining buffer set (cat. no. 00-5523-00) according to manufacturer protocol and stained with PE/Cy7 anti-Foxp3 (1:50, Thermo, 25-5773-82), PE anti-IL-17F (1:50, Biolegend, 517007), APC anti-LAP (TGFb1; 1:50, Biolegend, 141405). FACS data were collected by BD FACS Diva Software. The analysis was done using FlowJoTM 10. For the isolation of brain microglia and neutrophils, brains were extracted removed from the skull and homogenized to form a single cell suspension, then resuspended and centrifuged in a 30% Percoll Plus (GE Healthcare, 17-5445-02) gradient in HBSS at 800 g, 25°C, for 25 min with an acceleration of 4 and a deceleration of 3. Cells were pelleted and stained with rat APC anti-mouse Fcrls (1:400, clone 4G11, Butovsky Lab)51 and all antibodies listed above. After washing, CD115–CD11b+Ly6cintLy6g+ neutrophils and Ly6c–CD11b+Fcrls+ microglia were sorted using BD FACSAriaTM II (BD Bioscience). RNA libraries were prepared for sequencing using standard Illumina protocols