Fitc rat anti mouse cd11b

Vitamin C (purity: > 99.9%) was purchased from Shandong Xinhua pharmaceutical CO., ltd. DMEM Medium, Fetal Bovine Serum (FBS), Wright-Giemsa staining, and Albumin Bovine V were purchased from MACGENE. RBC lysis Buffer, Sulforhodamine B (SRB), RNase A, Propidium Iodide (PI), and rat IgG were purchased from Solarbio. Triton X-100 solution, Ca²⁺ specific fluorescent probe Fluo-4/AM, JC-1 Staining Kit, One Step TUNEL Apoptosis Assay Kit, and ATP Determination Kit were purchased from Beyotime. FITC Rat Anti-Mouse CD11b was purchased from BD Biosciences. APC Rat Anti-Mouse CD206 was purchased from Miltenyi Biotec. Western blot Antibody Diluent was purchased from Epizyme. Western Bright™ ECL and Western Bright™ Peroxide were purchased from Advansta. Goat Anti-Rabbit IgG (H+L) HRP was purchased from Affinity Biosciences. Vimentin (5G3F10) Mouse mAb, Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor^® 488 Conjugate) and β-actin (13E5) Rabbit mAb were purchased from Cell Signaling Technology. E-cadherin Rabbit PolyAb was purchased from Proteintech.

The RAW264.7 cells were harvested from a 6-well plate and washed with cold PBS three times. Then, the cells were stained with PE Rat Anti-Mouse F4/80 (565410, BD, United States), FITC Rat Anti-Mouse CD11b (557396, BD, United States), APC-Cy7 Hamster Anti-Mouse CD11c (561241, BD, United States), PE-Cy7 Rat Anti-Mouse CD16/CD32 (560829, BD, United States) and Rat Anti-Mouse CD206 (565250, BD, United States) antibodies at 4°C for 30 min. After staining, the cells were washed with PBS three times and resuspended with 100 μL of PBS for FACS analysis using a BD FACSAria II instrument (BD, United States). The data were further analyzed using Flowjo software 7.6.

All cell culture media, trypsin, fetal bovine serum (FBS), and antibiotic/antimycotic solutions were obtained from Invitrogen (Carlsbad, CA, USA). The L-929 fibroblast (CCl-1), LADMAC macrophage/monocyte (CRL-2420), and C3H/HeJ mouse I-13.35 splenic cell lines (CRL-2471) deficient for TLR-4 were purchased from the American Type Tissue Collection (ATCC) (Manassas, VA, USA). Endotoxin tested (less than 0.1 ng/μg) IFNγ (#I1000) was purchased from US Biological (Salem, MA, USA), the inducible nitric oxide synthase (iNOS) (#2977) antibody from Cell Signaling Technology (Danvers, MA, USA), the anti-monocyte/macrophage antibody (MOMA-2, ab33451) and anti-integrin beta-1 antibody (CD29) (#ab23834) from Abcam (Cambridge, MA, USA), and the fluorescein isothiocyanate (FITC) rat anti-mouse CD44 (#553133), phycoerythrin (PE) rat anti-mouse CD105 (#562759), PE rat anti-mouse Ly-6AE (Sca-1) (#561076), FITC rat anti-mouse CD45 (#553080), FITC rat anti-mouse CD106 (#553332), PE rat anti-mouse CD73 (#557041), and FITC rat anti-mouse CD11b (#553310) were purchased from BD Biosciences (San Jose, CA, USA). Human ox-LDL was purchased from Intracel (Frederick, MD, USA). Gamma-irradiated LPS from Escherichia coli (#L4391) and all other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified.

Retinas and RPE-Choroid from both WT and p75^NTR KO mice, with or without laser, were obtained 4 days after the lesion. Under dissecting microscope, retinas and RPE-Choroid were collected in separate tubes and homogenized by gentle pipetting in FACS buffer (cold PBS with 2% FBS, 0.1% sodium azide). Cell suspensions were filtered through a 70 µm cell strainer and washed in FACS buffer. Viability of the cells was checked by Alexa Fluor 700 NHS ester dye (1/15.000; Molecular Probes, Eugene, OR, USA) [37 (link)]. Cells were incubated for 30 min at 4 °C with mouse anti f4/80 (1/50; Invitrogen), rabbit anti CX3CR1 (1/100; Abcam, ab8021), FITC rat anti mouse CD11b (1/200; BD Biosciences, Franklin Lakes, NJ, USA), and APC e-Fluor 780 anti-mouse CD45 (1/200; Bioscience). Then, cells were washed with FACS buffer and incubated with goat against mouse IgG conjugated with Alexa Fluor 594 (1/250; Molecular Probes, Eugene, OR, USA) for 30 min at 4°C. FACS of at least 350,000 cells from each RPE-Choroid and 1,000,000 cells from each retina was performed. The labeled cells were analyzed on a BD LSR Fortessa X-20 cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with the FlowJo software (TreeStar), and data are analyzed using FlowJo software (version 7.6.5; FlowJo, Ashland, OR, USA).