Sanne de Bruin

149 Storage of RBCs in PAGGGM improves metabolism after transfusion but has no effect on PTR USA, product code: IC27671P, 1:10), GAS-6 (R&D systems, Minneapolis, USA, product code: MAB885, 1:5), Protein-S (Abcam, Camebridge UK, product code: ab61364, 1:5), Anti complement iC3b (Quidel, San Diego, USA, product code: A209, 1:5), Wheat germ agglutinin (conjugate alexa 488, Invitrogen, Carlsbad, USA, product code:W11261, 1:500), Anti-thrombospondin (A6.1) (Thermo Scientific, Landsmeer, The Netherlands, product code: MA5-13398, 1:50), CR1 (rabbit polyclonal; kind gift fromProf. Dr. M. R. Daha, Leiden University Medical Center, the Netherlands, 1:500), Bcam (R&D systems, productcode AF148, 1:100), C3-19 (a kind gift from Diana Wouters, Department of Immunopatholo- gy, Sanquin, Amsterdam, The Netherlands, 1:100), 4N1K (4N1K, amino acid sequence; KRFYVVMWKK, 1:100), 4NGG (amino acid sequence; KRFYGGMWKK, 1:100), Streptavidin (conjugate alexa 488/647, Invitrogen, Carlsbad, USA, product code: S-32354, 1:200), Mouse isotype control IgG1 (Invitrogen, Carlsbad, USA, product code: MG100, 1:5), Mouse isotype control IgG1 (conjugate alexa 488, Invitrogen, Carlsbad, USA, product code: MG120, 1:5), Mouse isotype control IgG2A (Invitrogen, Carlsbad, USA, product code: MG2a00, 1:5), Mouse isotype control IgG2A (conjugate alexa 488, Invitrogen, Carlsbad, USA, product code: MG2a20, 1:5), Mouse isotype control IgG2A (conjugate PE, Sanquin, 1:50), Rabbit isotype control IgG (Invitrogen, Carlsbad, USA, product code: 026102 , 1:200), Goat anti-human IgG (conjugate alexa 647, Invitrogen, Carlsbad, USA, product code: A-21445, 1:200), Goat anti-mouse IgG (conjugate alexa 488, Invitrogen, Carlsbad, USA, product code: A-11001, 1:200), Goat anti-rabbit (conjugate FITC, Invitrogen, Carls- bad, USA, product code: A11008, 1:200), Rabbit anti-goat (conjugate FITC, Invitrogen, Carlsbad, USA, product code: A11078, 1:200) Flow cytometry EDTA anticoagulated blood samples were analysed one day after sampling. The amount of bioRBCs was quantified by flow cytometry after staining with Streptavidin Alexa Fluor 647 conjugate (Thermo Fisher Scientific, catalogue number: S32357). The PTR of bioRBC was calculated with respect to the percentage bioRBCs present in the circulation 10min- utes after transfusion. The PTR was assessed until 90 days after transfusion. To assess the clearance of RBCs in the first 10minutes, the distribution of fresh and stored bioRBCs in the mixed RBC concentrate prior to infusion was compared with the distribution of fresh and stored RBCs recovered from the circulation after transfusion. A difference in distribution indicates that one of the populations is faster cleared from the circulation. To investigate the role of several “eat me” and “don’t eat me” markers double and triple staining was used to quantify the clearance markers separately in the different popu- lations. Membrane markers were expressed as percentage of fluorescent positive cells or as mean fluorescent intensity (MFI) per population. 6