Magnetic activated cell sorting technology

Mouse renal proximal tubule cells (mRPTCs) were isolated from BALB/c mice kidneys using the Miltenyi Biotec's Magnetic‐Activated Cell Sorting technology. We used biotinylated Lotus tetragonolobus lectin (Vector #B‐1325) for the isolation of mRPTCs. The purity of the isolated cells was assessed by immunofluorescence staining using an antibody to SGLT2 (a marker protein of S1 and S2 segments of renal proximal tubule), as used previously.22 mRPTCs from C5Bl/6J mice were cultured from progenitor kidney cells isolated from mouse embryo kidneys of C57Bl/6J mice (kindly supplied by Dr. Ulrich Hopfer, Case Western Reserve University, School of Medicine), as reported by Woost et al.23

CD133+CXCR4+ EC cells were separated by Magnetic-Activated Cell Sorting technology (MACS; Miltenyi Biotec). Cultured EC cells at 70%–85% confluence were trypsinized by 0.25% trypsin and resuspended in cold (2–8 °C) MACS buffer (PBS [pH 7.2], 0.5% BSA, and 2 mM EDTA). Cells were stained with the CD133 FITC-conjugated primary antibody (Miltenyi Biotec) according to the manufacturer's recommendations. CD133 FITC-conjugated antibody was added per 10⁷ total cells. The cells were then resuspended and 20 μL anti-FITC Multisort microbeads and 80 μL buffer were added per 10⁷ total cells, mixed well, and incubated 15 min at 2–8 °C. The cells were then washed, applied to a column placed in the magnetic field of the MACS Separator (Miltenyi Biotec). For the second magnetic labeling and separation, 20 μL of MultiSort Release Reagent was added per 1 mL of cell suspension, Next, the MACS Micro Beads were added to magnetically label the cells for the second marker, the CXCR4 PE conjugated antibody (Beckman coulter, USA). The reaction was mixed well and incubated according to the manufacturer's recommendations.