Anti cd71 apc

Cells were washed twice in PBS and pelleted in a 96- well plate. For the conjugated antibodies cells were stained in 50 μl of flow buffer (0.1% BSA in 1 × PBS) for 30 min at 4 °C in the dark. Cells were washed twice in flow buffer and resuspended in 100 μl of flow buffer prior to reading. We used the following antibodies at the dilutions indicated by the manufacturer: anti-CD49d PE-Violet 770 (Miltenyi Biotec), anti-CD36 Violet Blue (Miltenyi Biotec), anti-CD71 APC (Miltenyi Biotec), anti-basigin FITC (Invitrogen), anti-CD44 APC (Miltenyi Biotec), anti-CD55 APC (Miltenyi Biotec). Anti-AQP1 PE (B-11; Santa Cruz Biotechnology) was used at a 1:100 dilution. To measure ART4 expression, cells were stained with anti-DO3 eluate at a 1:2 dilution for 30 min at 4 °C in the dark and recognized with anti-human IgG Alexa Fluor 647 (life technologies) at a 1:200 dilution for 1 h 4 °C in the dark. Cells were analyzed by using a MACSQuant analyzer 10 flow cytometer (Miltenyi Biotec) and data were analyzed by using the software FlowJo, version 10.4. 50,000 cells were acquired for each sample and cell populations were separated by a live/dead Propidium iodide stain.

Cells were incubated with antibodies for cell surface markers including anti-GlyA-APC/Vio770, anti-CD36-PE, and anti-CD71-APC (1:20, 130100268, 130095472, and 130091727, respectively, all from Miltenyi Biotech, Germany). For intracellular staining, cells were fixed with 4% paraformaldehyde and resuspended in permeabilization buffer (Merck, US). Fixed cells were then incubated with anti-FoxO3 antibody (1:400, 12829, Cell Signaling Technology, US) for 1 h. After washing, cells were stained with FITC-conjugated secondary antibody (1:100, AP307F, Merck, US) for 30 min, and finally counter-stained with propidium iodide (1:50, Miltenyi Biotech, Germany) for 10 min. All incubation steps were performed at 4°C. Cells were analyzed using a Flowsight imaging flow cytometer (Merck, US) and images were analyzed with IDEAS 6.2 analysis software.
To determine levels of apoptosis following staining with cell surface markers, cells were incubated with Annexin V-FITC (1:50, Biolegend, US) and fluorescent intensities were measured using imaging flow cytometry. For the detection of ROS formation, cells were incubated for 30 minutes at 37°C with 10 μM CellROX green reagent (Invitrogen, US). Thereafter, cells were stained for cell surface markers, and analyzed using imaging flow cytometry.