Hamster igg

Antibodies with the following specificities used for flow cytometry and histology were purchased from BD Bioscience, eBioscience or Biolegend: CD45.2-biotin, Lamda Light chain-biotin, GFP- Alexa Fluor 488 (AL488), rabbit IgG-PE, CD38-PE-Cy7, CD95-PE-Cy7, BCL6-Al647, Streptavidin-Al700 or -Brilliant Violet 421 (BV421), CD45RA (B220)-allophycocyanin (APC)-Cy7, IRF4-eFluor 450. RelB (C19) was from Santa Cruz Biotech, rabbit IgG Fab₂-Al555 was from Cell Signaling Technologies. Purified αCD40 (FGK4.5) (RRID: AB_2490239) and αCD40L (MR1) (RRID: AB_1612465) were purchased from BioXCell. AFRC-Mac-1 cell (glycoprotein of dog chlamydomonas) (RRID: CVCL_K178) (Sigma-Aldrich) produced Rat IgG2a istotype control was purified from culture supernatants by affinity chromatography, using a staphylococcal protein G column (ThermoFisher Scientific) and filter sterilized. Hamster IgG was from Jackson ImmunoResearch Laboratories.

Brains and spinal cords from transgenic GFAP-EGFP mice were dissociated using the papain-based neural tissue dissociation kit (Miltenyi Biotec, Germany). Myelin was separated from the cells on a discontinuous Percoll gradient (GE Healthcare Biosciences AB, Uppsala, Sweden) and the cells were washed and incubated with blocking solution containing HBSS, FBS (Sigma-Aldrich), anti-Fc receptor antibody (BD Biosciences, Brøndby, Denmark), hamster IgG (Jackson ImmunoResearch, West Grove, PA, USA), and sodium azide. The cells were then labeled with phycoerythrin (PE)-conjugated anti-CD45 (BD Biosciences) for 15 min at 4 °C and propidium iodide (PI, Sigma-Aldrich) to detect microglia/macrophages and non-viable cells, respectively.
Cells were sorted using a FACSVantage SE DiVa cell sorter (BD Biosciences). Astrocytes were defined as EGFP positive and CD45 negative (Fig. 6b). Microglia were defined as EGFP negative and CD45^dim. Sorted astrocytes and microglia were re-analyzed by flow cytometry and quantitative real-time RT-PCR to verify purity.