Red blood cell isolation and flow cytometry

Peripheral blood from the hospitalized COVID-19 patients and healthy controls was collected in ethylenediaminetetraacetic acid tubes and processed within 24 hours. The blood samples were layered carefully with one volume of Ficoll-Paque (GE Healthcare, Little, Chalfont, Buckinghamshire, UK), centrifuged at 2000 rpm for 20 minutes with the brake off at 4°C. Peripheral blood mononuclear cells were isolated from whole blood by density centrifugation using the Ficoll-Paque gradient and kept for other studies. The pellet supernatant (RBCs) was stained with different surface markers. Firstly, 1 μL of RBCs was washed with phosphate buffer saline (PBS) and stained with anti CD235a (phycoerythrin), CD36 (allophycocyanin) and CD71 (fluorescein isothiocyanate) for 30 minutes in the dark at a 1:100 ratio. The pellet cells were then washed with PBS and resuspended in 200μl of PBS. The samples were analyzed using a BD FACSCanto II system (BD Bioscience, San Jose, CA, USA) and FACSDiva software, version 6 (BD Biosciences). The RBCs were gated based on side scatter and forward scatter. The data were analyzed using FlowJo software version 7.10 (Tree Star, Ashland, Oregon, USA).