Cd133 cell isolation kit

Detached cells (1 × 10⁷) were incubated with the microbead‐conjugated CD133 antibodies before being magnetically separated using a CD133 Cell Isolation Kit (Miltenyi Biotec). The collected CD133⁺ cells were next incubated by the microbead‐conjugated CD44 antibodies and then subjected to magnetic separation using a CD44 Cell Isolation Kit (Miltenyi Biotec) to isolate the CD133⁺/CD44⁺ cells.

After 4 days of culture, CD133⁺ fraction was recovered by immunomagnetic separation of total renal cells using CD133 Cell Isolation Kit (Miltenyi)62 (link). Clones were generated from CD133⁺ cells by limiting dilution in 96-well plates and immunofluorescence and FACs analysis were used to identify clones positive for CD24 (Santa Cruz Biotechnology) and CD106 (Sigma-Aldrich) (Figure S5A–C). One CD133⁺ CD24⁺ CD106⁺ clone was then characterized for the ability to differentiate toward podocytes, and used for all in vitro experiments. Podocyte differentiation was obtained by culturing cloned PECs for 6 and 24 hours with VRAD medium18 (link). PECs exposed to the VRAD medium exhibited a significant increase in the podocyte markers CD2AP and NEPHRIN, as determined by RT-qPCR (Figure S5D). Immunofluorescence analysis confirmed that PECs exposed to VRAD medium were positive for α-actinin-4, CD2AP and nephrin (Figure S5E).

Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were obtained from HyClone (Logan, UT, USA). Epidermal growth factor, basic fibroblast growth factor (bFGF), and leukemia inhibitory factor (LIF) were obtained from Peprotech (Rocky Hill, NJ, USA). The B27 (1×) serum-free supplement was purchased from Gibco Life Technologies (Grand Island, NY, USA). Nestin, GFAP, and β-tubulin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). FITC-conjugated IgG and related secondary antibodies were obtained from Boshide Co., Ltd. (Wuhan, China). The anti-CD133 antibody, its buffer solution and the CD133 cell isolation kit (magnetic activated cell sorting method) were purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany). The cell counting kit 8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan). SYBR Green I fluorochrome was purchased from Biotium, Inc. (Hayward, CA, USA).

Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham's (DMEM/F12) with high glucose medium was purchased from GE Healthcare (HyClone; Logan, UT, USA). Fetal bovine serum (FBS), trypsin, streptomycin, benzopenicillin and B-27 (1X) serum-free Supplement were purchased from Gibco Life Technologies (Grand Island, NY, USA). Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) were obtained from Peprotech (Rocky Hill, NJ, USA). A CD133 cell isolation kit (magnetic-activated cell sorting method) was purchased from Miltenyi Biotec GmbH (Bergisch Gladbach, Germany), while the lentivirus was provided by Shanghai GeneChem Co., Ltd. (Shanghai, China). The Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan), while temozolomide (TMZ) was purchased from Tasly Pharmaceutical Co., Ltd. (Tianjin, China). SYBR Green I fluorochrome was purchased from Biotium, Inc. (Hayward, CA, USA) and MMLV Reverse Transcriptase was purchased from Aidlab Biotechnologies, Co., Ltd. (Beijing, China).

C6 and U87 glioblastoma parental cells were trypsinized and suspended with ice-cold phosphate-buffered saline (PBS), centrifuged at 800 g for 5 min, and then resuspended in 1 × PBS with 0.5% bovine serum albumin (BSA) and 2 mM EDTA. Magnetically labeled anti-CD133 antibody from the Miltenyi Biotec CD133 cell isolation kit was used to isolate glioma CD133⁺ cells, as previously described [13 (link)]. CD133-PE conjugated antibody was applied for cell staining and evaluating the efficiency of magnetic separation via flow cytometry. The cell suspension was then placed within an autoMACS separator for magnetic separation. Labeled cells migrated toward the magnet; the unlabeled cells in suspension were drawn off. The remaining (labeled) cells were resuspended and then returned to the separator for further separation. The magnetic separation procedure was repeated twice to increase the efficiency of the magnetic separation. After the final elusion of the positive fraction of interest, the harvested cells suspended in culture medium were allowed for the downstream application. The separation purity was conducted via flow cytometry with a FACSCalibur machine (BD Biosciences).

CD133+ cells were isolated from primary surgical GBM biopsy specimens in accordance with protocols approved by the Fudan University Institutional Review Broads. All patients have been informed and consented to involve in this study. CD133+ and CD133‐ cells were isolated through magnetic cell sorting with CD133 cell isolation Kit (Miltenyi Biotec, cat#130‐100‐857) as previously described.^[2]

GSCs were isolated from surgical human GBM tissues in accordance with protocols approved by the Fudan University Institutional Review Broads. Briefly, tumor specimens were washed and enzymatically dissociated into single cells. The isolated tumor cells were briefly placed in serum‐free Dulbecco's modified Eagle's and F12 media (DMEM/F12) supplemented with B27 lacking Vitamin A (Invitrogen). GSCs and DGCs were separated through magnetic cell sorting with CD133 Cell Isolation Kit (Miltenyi Biotec). Magnetic separation was performed at least twice. To ectopic expression of SLC1A5‐FLAG in GSCs, GSCs were infected with lentivirus in the presence of 8 µg ml⁻¹ polybrene (Sigma‐Aldrich).