Anti cd11b

Lateral heads of the gastrocnemius muscles were enzymatically digested; cell suspensions were depleted of neutrophils, T-cells, and B-cells by MACS negative selection with anti-Ly6G, anti-CD3, and anti-CD19 (Biolegend and Miltenyi Biotec); and macrophages were isolated by MACS positive selection with anti-CD11b (Miltenyi Biotec). Thus, macrophages were obtained as the CD11b-positive, Ly6G/CD3/CD19-negative fraction.

Single-cell suspensions of spleens and LNs (axillary, inguinal and mesenteric lymph nodes) were used to isolate CD4⁺ T or CD4⁺CD25⁻ T cells by negative selection, respectively, using CD4⁺ T cell or CD4⁺CD25⁺ regulatory T cell isolation kit for magnetic separation (Miltenyi Biotec, Bergisch Gladbach, Germany), achieving >92% purity as determined by FCM analysis.
To purify CD11b⁺ Mφ, splenic mononuclear cells were isolated using Percoll density-gradient centrifugation as described previously,^{44 (link)} and then incubated with anti-CD11b (Miltenyi Biotec) according to the manufacturer’s instructions. The purity of the isolated CD11b⁺ Mφ was >95%.

Kidneys were processed as previously described (22 (link)) and the cell pellet was resuspended in PEB buffer (0.5% BSA, 2mM EDTA in PBS pH 7.4) containing anti-mouse CD16 (BD Pharmingen San Diego, CA). Anti-CD11c coated MACS beads (Miltenyi Biotech – Bergisch-Gladbach, Germany) 10ul/10⁷ cells were added for 15 minutes followed by anti-CD11b coated beads for 15 minutes. Cells were then stained with anti-F4/80-PE (Invitrogen, Carlsbad, CA), anti-CD11c-FITC (BD Pharmingen) and anti-CD11b-APC (eBiosciences, San Diego, CA) and loaded into the AutoMACS. Separation of the labeled cells was performed using either POSSEL_S or POSSEL_D program.
The positive fraction was further fractionated by cell sorting as shown in Figure 1 and the purity of the populations was evaluated by post sort analysis. Populations were further characterized by staining with anti-mouse CD103-PerCP-Cy5.5, BTLA-PE, 33D1-biotin, F4/80-Pacific blue (Biolegend, San Diego, CA), CD8α-PerCP, MHCII-AF700, CD14-PerCp-Cy5.5 (eBiosciences), CD43-PE, CD62L-PE, MHCI-FITC (BD Pharmingen), DEC205-PE (Miltenyi Biotech) and VLA4-FITC (Southern Biotech, Birmingham, Alabama) (20 (link), 22 (link)).

Cx3cr1^gfp/+ iPSC were differentiated into microglia using a two-stage protocol. In the first stage, a modified protocol for the hematopoietic differentiation of iPSC^{40 (link)}, Cx3cr1^gfp/+ iPSC colonies were dissociated into a single-cell suspension using 1 mg/mL collagenase (Invitrogen) and Accutase (Stemcell Technologies) and plated at a density of 1.5e6 cells per 15-cm tissue culture dish of confluent OP9 stromal cells. This Stage 1 culture was maintained in alpha-MEM with 10% defined FBS (Hyclone), 2-mercaptoethanol, and penicillin/streptomycin for seven days. On day four, the media was fully replaced. After seven days of Stage 1 culture, cells were harvested by dissociation with trypsin (Gibco) and transferred onto a confluent astrocyte monolayer for Stage 2 culture. Stage 2 cultures were maintained in DMEM/F12 media (Gibco) with 10% defined FBS, 2 mM 2-mercaptoethanol, 2 mM Glutamax (Gibco), non-essential amino acids (Gibco), 20 ng/mL recombinant murine GM-CSF (Peprotech), 20 ng/mL recombinant murine M-CSF (Peprotech), 20 ng/mL recombinant murine IL-3 (Peprotech), and penicillin/streptomycin. Media in Stage 2 cultures was replaced twice weekly. Purification of iPS-MG from Stage 2 cultures was carried out using anti-CD11b or anti-CD39 magnetic beads (Miltenyi) according to manufacturer’s instructions followed by optional FACS sorting of iPS-MG by gating on GFP⁺ cells.