Lsrii sorp

Mice were euthanized and eyes were immediately enucleated. Retinas, optic nerves, and spleens were dissected in ice-cold, filter sterilized HBSS (Gibco; 14175-095) and placed in HBSS with dispase (5 U/ml) (Stemcell Technologies), DNase I (2000 U/ml) (Worthington Biochemical), and SUPERase (1 U/μl) (ThermoFisher Scientific). The tissues were shaken at 350 rpm for 60 min at 37 °C in an Eppendorf Thermomixer R and then titrated with a 200 μl pipette to dissociate cells. Cells were centrifuged at ~ 3000 g for 5 min and suspended in a new solution by titration. Ovomucoid trypsin inhibitors (10 mg/ml) were added to the 2% BSA in HBSS block solution to inhibit proteases. Samples were kept on ice and protected from light for blocking and antibody incubations. Primary antibody solution contained anti-Cd11b, anti-CD45, anti-Cd11c, and DAPI. Samples were blocked for 1 h, incubated with primary antibodies in block solution for 2 h, washed 3×, incubated in secondary antibodies for 1 h, washed 3×, and then suspended in block solution for flow cytometry on BD Biosciences LSR II SORP. Tissue collected from the spleen and processed the same was used to guide analysis of the myeloid cell populations.

Primary mixed cortical cultures of glial cells from 3-day-old pups were generated and microglia were fluorescently labeled and sorted as previously described [18 (link)]. In brief, 17 days after plating, cultures were dissociated (HyClone Trypsin .25%; Thermo Scientific) and resuspended in FACS buffer: HBSS (Gibco; Invitrogen 14025) supplemented with 2% BSA (Sigma-Aldrich, A7906) and containing 1 U/μl SUPERase In™ RNase Inhibitor (Ambion; Life Technologies, AM2694). Cells were centrifuged at 1305 g for 5 min and suspended in 50 μl of fresh FACS buffer to wash. The cells were stained for 1 h at 4 °C with chicken anti-GFAP (Abcam, ab4674) to label astrocytes and rabbit anti-IBA1 (Wako, 016-20001) to label microglial cells. Cells were washed three times and incubated for 30 min at 4 °C with secondary antibodies: donkey anti-rabbit 647 (Invitrogen, A31573) and goat anti-chicken 488 (Invitrogen, A11039). Samples were re-suspended in 200 μl of FACS buffer and sorted on BD Biosciences LSR II SORP. Purified microglia were collected separately and stored in RLT Buffer (QIAGEN, 79216) at − 80 °C. Total RNA was isolated (QIAGEN, 74104) from purified samples from D2.C3ar1^−/− and D2.C3ar1^+/+ mice.

Bone marrow cells flushed from long bones were syringed five times through a 23-G needle and filtered through a 100μm nylon cell strainer. Red blood cells were lysed with 0.15 M NH₄Cl. Cells were resuspended in PBS and filtered through a 70μm strainer. Samples were incubated with Fixable Viability Dye eFluor780 (eBioscience, San Diego, CA) and Fc receptors were blocked with anti-mouse CD16/CD32 (Biolegend, San Diego, CA) at a dilution of 1:100. Cells were then incubated with anti-mouse CD115 APC, anti-mouse CD11b PERCP-Cy5.5, anti-mouse B220 Pe-Cy7, anti-mouse CD11c PE, and anti-mouse Gr-1 Brilliant Violet 421 (BioLegend) at a dilution of 1:100 in PBS/2% FBS. Samples were acquired on a FACSCanto II or LSR II SORP (BD Biosciences, San Jose, CA). Analysis was performed with FlowJo software (Tree Star, Ashland, OR).

Blood-isolated or tissue-isolated leukocytes were stained using the marker-specific fluorochrome-labeled antibodies indicated in Supplementary Table 2. For intracellular detection of proteins, including FOXP3 (murine and human), IL-17A, Helios, Ki67, and GZMB, a FOXP3 Transcription Factor Staining Buffer Kit was used (ThermoFisher). For the assessment of intracellular IL-17A or IFNγ, cells were stimulated with 1 µg/ml ionomycin and 10 ng/ml phorbol 12-myristate 13-acetate (PMA) in the presence of 5 µg/ml Brefeldin A (all Sigma-Aldrich). After staining with anti-CD8 antibodies, the BD Cytofix/Cytoperm Kit (BD Biosciences) was used for cell fixation and permabilization.
Samples were acquired using LSRII SORP, FACSCanto, or FACSCelesta instruments (all BD Biosciences) and analyzed using a DIVA (all BD Biosciences) or FlowJo (version 7.2.5, Tree Star) software.

Cells were harvested and adjusted with fresh medium to the cell density of 1 × 10⁶ cells per ml. Aliquots thereof were put aside for control purpose, and either Verapamil (20–100 μM) (Catalog number V4629), or reserpine (20–100 μM) (Catalog no. 83580) was added. Verapamil and reserpine are known to block several ABC drug transporters (Sigma-Aldrich). Cells are distinguished from debris on the flow-cytometric profile based on the forward scatter and side scatter (SSC). Cell doublets and aggregates are gated out based on their properties displayed on the SSC area versus height dot plot. Side population (SP) cells are recognized as a dim tail extending toward the lower “Hoechst Blue” signal. The gating tree indicates the sequential procedure applied to select out the final population for SP discrimination and the percentage of cells (gated events) resulting from each gating step. The gating strategy are represented as shown in Supplementary Fig. 8. Cells were filtered using 35 μm filter round-bottom FACS tubes (BD Biosciences) immediately before data acquisition on either an LSR II SORP or Fortessa (BD) and data analyzed using FlowJo software (Tree Star, Inc.).