Gallios flow cytometer

Tumor tissues were minced and incubated with collagenase type II (1 mg/ml, Thermo Fisher) at 37°C for 40 min to obtain a single-cell suspension. For the staining of extracellular target proteins, 1 × 10⁶ cells were first incubated in a mixture of PBS, 1% FBS, and anti-CD16/32 antibody (BioLegend, #101301) to block non-specific binding and then labeled with the indicated antibodies at room temperature for 30 min. Fluorescence-activated cell sorting (FACS) was performed using a Beckman Coulter Gallios flow cytometer (USA), and the results were analyzed using FlowJo software version 10.0.7 (TreeStar). The following fluorochrome-coupled antibodies were used in the experiment: anti-CD45 (#103115), anti-CD206 (#141711), anti-CD3 (#100235), anti-CD4 (#100203), anti-CD8 (#100733), anti-NK1.1 (#108709), and anti-Gr1 (#108425) (all from BioLegend); and anti-CD11b (#12-0112), anti-F4/80 (#11-4801), anti-CD11c (25–0114), anti-MHCII (#17-5321), and anti-CD86 (#17-0862) (all from eBioscience) antibodies; the fixable viability dye eFluor 506 was from eBioscience (#65-0866).

Lipid peroxidation was assessed using a Click-iT lipid peroxidation imaging kit (ThermoFisher Scientific; Rockford, IL, USA). 661W photoreceptor cells seeded on 15-mm glass slides in a 24-well plate were pretreated with 50-μM Click-it LAA for 30 min at 37 °C and then incubated with 5-μM atRAL for 3 and 6 h, 100-μM cumene hydroperoxide (CH) for 6 h, or vehicle (DMSO) alone for 6 h at 37 °C. Alternatively, cells were pretreated with 20-μM Fer-1 for 2 h at 37 °C and incubated with 50-μM Click-it LAA for 30 min, followed by treatment with or without 5-μM atRAL for 6 h, respectively. Cells treated with atRAL or vehicle (DMSO) alone served as controls. Subsequently, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed three times with PBS, permeabilized by a 0.5% Triton X-100, and then blocked with 1% BSA. After 50-μl Click-iT reaction cocktail containing 5-μM Alexa Fluor 488 was added into each slide, the cells were incubated for 30 min at 37 °C in the dark, stained with DAPI, and then examined by using an Olympus FV1000 confocal microscope (Tochigi, Japan). To further quantify lipid peroxidation, levels of lipid peroxides were evaluated by flow cytometry using C11-BODIPY dye (74 (link)). Samples were subjected to a Beckman Coulter Gallios flow cytometer (Brea, CA, USA), and data were analyzed using FlowJo 10 software (TreeStar; Ashland, OR, USA).

To explore the cell proliferation response in MLR, we used bromodeoxyuridine (BrdU) incorporation and then tested by flow cytometry. The protocol followed previous research. Additionally, peripheral blood of rat recipients at 7 days after transplantation was harvested and stained with APC-labeled anti-CD3, FITC-labeled anti-CD4 and PerCP-eFluor710-labeled anti-CD8 antibodies. Flow cytometry was determined by a Gallios flow cytometer and analyzed with FlowJo Software (Tree Star, OR).