Pe cy7 rat anti mouse cd45

Cell flow cytometry was performed using a NovoCyte® Flow Cytometer (ACEA Biosciences Inc., San Diego, CA, USA) according to the manufacturer's instructions. Briefly, ADSCs (1 × 10⁵ cells) were mixed into 0.5 ml of Perfusion Solution (CORNING, Manassas, VA, USA). Each antibody (1/100 of the volume) was added to the cell mixture and incubated on ice for 30 minutes. After washing the cells with Brilliant Stain Buffer (BD Biosciences, Franklin Lakes, NJ, USA), fluorescence-activated cell sorting (FACS) measurements were conducted. The following primary antibodies were used: Brilliant Violet 421™ Rat Anti-Mouse CD44 (BD Biosciences), Fluorescein Isothiocyanate (FITC) Rat Anti-Mouse CD90.2 (BD Biosciences), PerCP/Cy5.5 Anti-Mouse CD34 (BioLegend, San Diego, CA, USA), and PE/Cy7 Rat Anti-Mouse CD45 (BD Biosciences). Isotype-identical antibodies were used as controls.

For quantification of immune cells in the mouse RPE and retina, mouse retina and RPE-choroid were gently dissected and digested with 1.5 mg/ml collagenase A (Sigma-Aldrich, 10103578001) and 0.4 mg/ml DNase 1 (Sigma-Aldrich, 11284932001) at 37 °C for 1 h. Single-cell suspensions were generated by pipetting the tissue to release cells and passing cells through 70 μm filters. Cells were blocked with 1% mouse serum (Thermo Fisher Scientific, 10410) and 1 μg/μl Fc blocker (BD Biosciences, 553142), followed by antibody staining including BV650 Rat Anti-Mouse CD11b, (BD Biosciences, 563402, M1/70), PE-Cy7 Rat anti-Mouse CD45 (BD Biosciences, 561868), FITC Rat anti-Mouse CCR2 (SC203G11) (BioLegend, 150607), APC Rat Anti-Mouse Ly-6G (BD Biosciences, 560599, 1A8) and BV421 Rat Anti-Mouse Ly-6C (BD Biosciences, 562727, Al-21) in brilliant stain buffer (BD Biosciences, 563794,) at a concentration of 1 μg/mL for 30 min at room temperature. Flow cytometry was used to estimate the number of immune cells in the RPE/retina.

Cell flow cytometry was performed using a NovoCyte Flow Cytometer (ACEA Biosciences, Inc., San Diego, CA, USA) according to the manufacturer’s instructions. Briefly, mAdMSCs (1 × 10⁵) were added to 0.5 mL of perfusion solution (Corning, Manassas, VA, USA). Antibodies (1/100 dilution) were added to the cell suspensions, which were then incubated on ice for 30 min. Fluorescence-activated cell sorting was performed after washing the cells with Brilliant Stain Buffer (BD Biosciences, Franklin Lakes, NJ, USA). The primary antibodies were as follows: Brilliant Violet 421TM Rat Anti-Mouse CD44 (BD Biosciences), Fluorescein Isothiocyanate (FITC) Rat Anti-Mouse CD90.2 (BD Biosciences), PerCP/Cy5.5 Anti-Mouse CD34 (Biolegend, San Diego, CA, USA), and PE/Cy7 Rat Anti-Mouse CD45 (BD Biosciences). Isotype-specific antibodies were used as controls. Flow cytometry was performed as previously described [29 ,42 (link),43 (link)].