Easysep mouse sca1 positive selection kit

After enzymatic digestion of the kidney, cells were seeded in complete medium consisting of α-MEM w/o ribonucleosides w/o nucleosides with glutamax (Invitrogen) supplemented with 10% heat-inactivated FCS (PAA) and 1% Penicillin, Streptomycin (Sigma-Aldrich).
For primary renal cell cultures, cell suspensions were seeded in one T75 cm² flask (Falcon) per mouse and washed twice the next day with DPBS. Adherent cells were grown in complete medium until sub-confluence (7 days) at 37 °C under 5% CO₂ in a humidified atmosphere. Cells were trypsinized, counted in trypan blue (Gibco) and phenotyped by flow cytometry. Isolation of Sca-1⁺ renal cells was performed at the first passage by Sca-1 positive selection using magnetic beads (EasySep^™ Mouse Sca-1 Positive Selection Kit, Stemcell technologies). CD73⁺ kMSCs (CD73⁺ sca-1⁺ , EpCAM⁻, CD45⁻, CD31⁻) and Sca-1⁺ epithelial cells (sca-1⁺, EpCAM⁺, CD45⁻, CD31⁻) were selected from the cultures by high-speed sorting (INFLUX, BD Biosciences). For experiments, each replicate corresponds to cells isolated from individual mice or cells pooled from two to three mice after cell subset selection. Biological tests were performed just after selection or after one passage.

HSCs were collected according to the manufacturer's protocol for the magnetic bead‐based EasySep Mouse SCA1 Positive Selection Kit (StemCell Technologies, Vancouver, British Columbia, Canada). Briefly, bone cells were collected from the femur and tibia of C57BL/6 mice and made into a suspension of 1×10⁶ cells mL⁻¹ in StemSpan Serum‐Free Expansion Medium (SFEM) (StemCell Technologies) containing human IL‐6 (100 ng mL⁻¹, StemCell Technologies, Canada), human fms‐like tyrosine kinase‐3 (Flt3) ligand (100 ng mL⁻¹, StemCell Technologies), murine stem‐cell factor (SCF) (50 ng mL⁻¹, StemCell Technologies), and human thrombopoietin (TPO) (20 ng mL⁻¹, PeproTech, Cranbury, New Jersey, USA). Mouse SCA1 PE Labeling Reagent (50 µL mL⁻¹, StemCell Technologies) was first added to the cell suspension, and the mixture was kept in the dark at room temperature for 15 min. Then, a PE Selection Cocktail (70 µL mL⁻¹, StemCell Technologies) was added to the mixture and kept in the dark at room temperature for 15 min. After vortexing, dextran RapidSphere (50 µL mL⁻¹, StemCell Technologies) was added to the mixture and stored at room temperature in the dark for 10 min. After the mixture was incubated in the magnet for 5 min, the supernatant was discarded and SFEM was added to resuspend the cells. This step was repeated three times to obtain HSCs, and the cells were used in passages 3–5.