Anti cd47

Altered membrane phospholipid asymmetry and RBC ageing was evaluated with Annexin-V (Life Technologies, Canada) and anti-CD47 (BioLegend, clone miap301) staining. Briefly, 1 μL of blood was collected in 1 mL of PBS, and centrifuged. Pellet was resuspended in Annexin binding buffer (Life Technologies), stained with FITC-labeled Annexin-V, PE-labeled rat anti-mouse CD47 monoclonal antibody and streptavidin-APC, and incubated at 20°C, in the dark for 15 min. Cells were washed and resuspended in PBS prior to FACS analysis of geometric mean of fluorescence intensity (gmeanFI) relative to Annexin-V and CD47 in streptavidin positive cells. Intra-erythrocytic ROS were measured using a modification of the Amer method [25 (link)]. Briefly, 2 x 10⁶ RBCs/mL were incubated at 37°C for 15 min in the dark with streptavidin-APC and 2’,7’-dichlorofluorescein diacetate (DCFDA; Sigma-Aldrich) dissolved in methanol. Cells were washed in PBS and analyzed for gmeanFI of 2’,7’-dichlorofluorescein (DCF) within streptavidin positive cells. Positive controls for Annexin-V and CD47 binding, and ROS measurement were achieved with a 15 min cells treatment with 50 μM hydrogen peroxide prior to washing and labeling.

Cells were harvested using TrypLE™ Express Enzyme (12604013, Gibco, Waltham, MA, USA) and fixed using a Cytofix/Cytoperm™ Fixation/Permeabilization Kit (554714, BD). The cells were incubated at 4 °C for 45 min with anti-CD47 (323110, BioLegend, San Diego, CA, USA), anti-CD26 (302705, BioLegend), anti-CD45 (557883, BioLegend), anti-CD14 (565283, BioLegend), and CD86 (564544, BioLegend) antibodies. Stained cells were analyzed using a flow cytometer (FACS Canto or FACS Aria III cell sorter, Becton Dickinson). Data acquisition and processing were performed using FACS Diva software (Becton Dickinson).