Easysep biotin positive selection kit 2

Peripheral blood from T127 carrying FVB mice was collected through orbital sinus of isoflurane anesthetized mice. Blood was collected with EDTA blood collection microtubes. After serum aspiration, blood was diluted with PBS and put through density centrifugation with Ficoll. After carefully extracting the layer of the mononuclear cells, cells were washed three times with PBS. CD11b positive cells were isolated by EasySep Mouse CD11b positive selection Kit II (STEMCELL, 18970). The CD11b positive cell pool was incubated with primary C5aR1 antibody at 3 μg/ml in PBS with 2% FBS. Secondary antibodies conjugated with Biotin were added 1.2 μg/ml in PBS with 2% FBS. Magnetic column based cell sorting was performed by following manufacture’s manual of EasySep Biotin Positive Selection Kit II (STEMCELL, 17683). The purity of positive cells was assessed with flow cytometry. 5*10⁵ C5aR1^hi cells in 100 μl selection buffer or only 100 μl selection buffer was transferred to FVB mouse 2 weeks after T22 transplantation through tail vein injection. Olaparib treatment started on the second day after C5aR1^hi cell transplantation with the same dose indicated above.