Absorbance plate reader

bEnd.3 cells (7,500 cells/well) seeded in 96-well plates (Corning, New York, NY, United States) were assessed for cell viability using the Cell Counting Kit-8 assay as per the manufacturer’s instructions (Dojindo Molecular Technologies, Rockville, MD, United States). Absorbance was measured at 450 nm using an absorbance plate reader (Molecular Devices, LLC, San Jose, CA, United States), and cell viability was adjusted to % of bEnd.3 control group (bEnd.3 cells with no RBC co-incubation).

Hemoglobin was measured using 2,7-diaminofluorene (Sigma-Aldrich, St. Louis, MO, United States) as described previously (Chang R. et al., 2018 (link)). bEnd.3 cells incubated with RBCs were washed with PBS for total cellular hemoglobin (hemoglobin from attached and engulfed RBCs) or quickly washed with distilled water three times for intracellular hemoglobin (hemoglobin from engulfed RBCs). Cells were lysed with urea lysis buffer (0.2 M Tris–HCl buffer containing 6 M urea, Thermo Fisher Scientific, Waltham, MA, United States) and mixed at a 1:1 ratio with 2,7-diaminofluorene reaction buffer (10 mg 2,7-diaminofluorene in 10 ml urea lysis buffer containing 9% acetic acid and 0.3% hydrogen peroxide). Absorbance was measured at 620 nm using an absorbance plate reader (Molecular Devices, LLC, San Jose, CA, United States). A set of hemoglobin standards (Lee BioSolutions, Maryland Heights, MO, United States) was used to calculate the amount of hemoglobin.