Ly6c fitc clone al 21

Red blood cells were lysed in bone marrow and collagenase IV (Sigma)-treated lung samples. Lung, spleen or BM cells (3 × 10⁶) were then stained with fixable viability dye eFluor501 (eBioscience) at the concentration of 1:1 000 for 30 minutes (4°C). Subsequently, the cells were washed with PBS supplemented with 0.5% BSA (Wisent) and incubated with anti-CD16/32 (clone 93, eBioscience) at a concentration of 1:100 in PBS/0.5% BSA at 4°C for 10. After washing, cells were incubated with fluorochrome tagged antibodies at 4°C for 30 minutes. Antibodies for the innate panel: anti-CD11b–Pacific Blue (clone M1/70, eBioscience), anti-CD11c–PE-Cy7 (clone HL3, BD Bioscience), Siglec-F–PE-CF594 (clone E50–2440, BD Bioscience), F4/80–APC (clone BM8, eBioscience), Ly6C–FITC (clone AL-21, BD Bioscience), Ly6G–PerCP-eFluor710 (clone 1A8, eBioscience). Antibodies for the adaptive panel: anti-CD3–PE (clone 145-2C11, eBioscience), anti-CD19–PE-Cy7 (clone eBio1D3 (1D3), eBioscience), anti-CD4–eFluor450 or anti-CD4-FITC (clone GK1.5, eBioscience), anti-CD8–AF700 (clone 53-6.7, BD Bioscience). All cells were subsequently washed with PBS/0.5% BSA and resuspended in 1% paraformaldehyde.

Control or clodronate encapsulated liposomes (Encapsula Nanosciences) were injected intravenously daily starting 1 day before NaIO₃ administration. Clodronate depletion of peripheral monocytes/macrophages was confirmed by flow cytometry. To deplete microglia, CX3CR1^CreER/+:Rosa26^iDTR/+and CX3CR1^CreER/+:Rosa26^+/+ mice (controls) were given five doses of 80 mg/kg of Tamoxifen (Sigma) in sesame oil by intraperitoneal injection (i.p.) for five consecutive days. Thirty days after the first Tamoxifen dosing, mice were given i.p. two doses of 2 ug diphtheria toxin (DT) (Sigma) for two consecutive days. Blood monocytes and retinal microglia were analyzed by FACS 6 days after the first DT dosing. Mice were injected with NaIO₃ on day 6 after the first DT dosing and photoreceptors were analyzed 3 days after NaIO₃ injection by FACS as described^{68 (link)}. Blood was collected by cardiac puncture and erythrocytes were removed by ACK lysis buffer (Life Technologies). Cells were resuspended in PBS/2% FBS/2 mM EDTA and Fc blocked (BD) and stained for CD115-APC (clone AFS98, eBiosciences) or Ly6C-FITC (clone AL-21, BD).