Sanne de Bruin

150 Chapter 6 All flow cytometry experiments were performed with a flow cytometer (BD FACS, BD Fortessa + HTS, BD Biosciences). The data were analysedwith computer software (FACS- Diva v.8.0.1, BD Biosciences). Glucose-6-phosphate dehydrogenase activity The G6PD activity was assessed per individual RBC using a flowcytometric assay as previously described 9,21 . In short, 10 µl RBC suspension was suspended in 90 µl PBS and 100 µl 0.125 mmol/l sodium nitrate (Sigma St. Lous, USA) and incubated for 20 minutes at room temperature for 20 minutes at a roller bench to convert all oxyhemoglobin into methemoglobin. RBCs were washed three times with PBS (200 RCF, 1 minute) and resuspended in 1 ml PBS. In the next step methemoglobin was reduced to oxyhemoglobin in a NADPH and thus G6PD dependent matter. To do so, the RBC suspension was incubated for 90 minutes at 37˚C with 100 µl PBS, 18 µl 0.28 mmol/L glucose (Sigma), and 6 µl 0.3mmol/L meth- ylene blue sulphate (Merck, Darmstadt Germany). After incubation 2.5 µl 400 mmol/L potassium cyanide (Sigma) was added and incubated at room temperature for 5 min- utes, followed by incubating 5 µl sample in 100 µl 1 mmol/L H 2 O 2 (Sigma) in PBS for 3 minutes. After incubation, the cells were stained with streptavidin alexa fluor 647 (In- vitrogen, S32357, 1:200) for 30 minutes at 4 degrees Celsius. Finally, the samples were washed twice (PBS, 1500 RCF 3 minutes) and analysed on the flowcytometer (BD FACS, BD Fortessa + HTS, BD Biosciences). Cell sorting procedure BioRBCs were isolated in two separate steps from the EDTA whole blood samples. This procedure is based on the differences in biotin density. In the first step, bioRBCs were isolated using magnetic beads, followed by the second step using flowcytometric cell sorting (supplemental Figure S1). Blood samples were washed twice to remove plasma and the buffy coat (SAGM, 1000 RCF for 15 minutes, 4˚C and 2500 RCF, 5 minutes, 4˚C). RBCs were incubated for 30min- utes at 0˚Cwith streptavidin-647 (1:100, Thermo fisher scientific) andwashed (2500 RCF, 5 minutes, 4˚C), followed by incubation for 15 minutes at 4˚C with 80 µl anti-Alexa fluor 647 micro beads (Milteny biotec). Incubated cells were flushed through an LS column (Milteny biotec) while put in amagnetic field. This columnwas flushed three times with a flushing buffer containing PBS (Fresenius Kabi), 10% tri sodiumcitrate (Merck) and 0.5% human serum albumin (Brocacef) to remove the unlabeled cells. The retained BioRBCs in the LS column were eluted in the flushing buffer after the column was removed from