Gentlemacs dissociator and tumor dissociation kit

Xenograft lines GBM12, GBM22, GBM39, and GBM59 and the isogenic TMZ-resistant xenograft lines GBM12-TMZ, GBM22-TMZ, GBM39-TMZ, and GBM59-TMZ were obtained from Jann N. Sarkaria at the Mayo Clinic. The xenograft lines were passed through mice as subcutaneous tumors and dissociated using a gentleMACS Dissociator and Tumor Dissociation Kit (Miltenyi Biotec Inc., #130–095-929) according the manufacturer’s protocol.
Cells were treated with FcR Blocking Reagent (20 μl/10⁷ cells, Miltenyi Biotec Inc., #130–059-901) for 10 min at 4 °C. Cells were then stained with mouse monoclonal anti-human IGF-IR fluorescein-conjugated antibody, clone #33255 (dilution 10 μl/10⁶ cells) or mouse IgG1 fluorescein-conjugated isotype control antibody (10 μl/10⁶ cells). After incubation for 20 min in the dark on ice, cells were washed three times with FACS buffer (PBS + 2% FBS) and then analyzed by flow cytometry with a BD Accuri™ C6 Cytometer (Becton Dickinson Biosciences). The percentage of IGF-1R⁺ and IGF-1R⁻ cells was determined with FlowJo software from Tree Star.

The tumor cells in a single cell suspension were isolated from the each xenograft within 2 hours by using the gentleMACs Dissociator and Tumor Dissociation Kit (Miltenyi Biotec Inc., Auburn, CA) according to the manufacturer’s guidelines. 0.5 × 10⁶ cells per sample for flow cytometry analysis were as follows: a) unstained; b) stained with mouse IgG1-PE/-FITC; c) stained with anti-human CD44-PE; d) stained with anti-human CD24-FITC; and e) stained with anti-human CD44-PE/CD24-FITC (Miltenyi Biotec Inc., Auburn, CA). The fluorescence intensity of these cell samples was analyzed by the Gallios flow cytometer (Beckman Coulter, Inc., Brea, CA). The ALDEFLUOR kit (Stemcell Technologies) was used for the identification of cancer stem cells from MDA-MB-231 xenografts by flow cytometry analysis.