Tumor Sphere Formation Assay

To test the ability of cells with modified gene expression levels to form tumor spheres, we generated tumor spheres as previously described [18 (link)]. In detail, the adherent growing cell lines were dissociated into single cells using trypsin/EDTA and 2000 single cells per well seeded in ultra-low attachment 6-well plates (Corning, NY, USA) using serum-free MEBM (Lonza, Basel, Switzerland) medium (SFM) supplemented with 1xB27 supplement (Gibco), 20 ng/ml human epidermal growth factor EGF (Peprotech, Hamburg, Germany), 10 ng/ml human basic fibroblast growth factor FGF (Peprotech), 20 IU/ml heparin (Baxter, Vienna, Austria), and 1% antibiotic/antimycotic solution (Thermo Fisher Scientific, containing 10,000 units/mL of penicillin; 10,000 μg/mL of streptomycin; and 25 μg/mL of Gibco Amphotericin B). After 14 days, the number of spheres was counted using a bright-field microscope in three independent replicates.

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Alkan H.F., Vesely P.W., Hackl H., Foßelteder J., Schmidt D.R., Vander Heiden M.G., Pichler M., Hoefler G, & Bogner-Strauss J.G. (2020). Deficiency of malate-aspartate shuttle component SLC25A12 induces pulmonary metastasis. Cancer & Metabolism, 8, 26.

Publication 2020

ultra-low attachment 6-well plates 1xB27 supplement human epidermal growth factor EGF human basic fibroblast growth factor FGF heparin antibiotic/antimycotic solution Amphotericin B

Amphotericin b Antibiotic Basic fibroblast growth factor Cell lines Cells Edta Epidermal growth factor Gene expression Heparin Human Microscope Penicillin Serum Streptomycin Supplement Trypsin Tumor

Corresponding Organization :

Other organizations : Massachusetts Institute of Technology, Graz University of Technology, Medical University of Graz, Dana-Farber Cancer Institute, Innsbruck Medical University, Universität Innsbruck, Harvard University, Beth Israel Deaconess Medical Center, The University of Texas MD Anderson Cancer Center, BioTechMed-Graz

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Variable analysis

independent variables

Gene expression levels

dependent variables

Tumor sphere formation

control variables

Dissociation of adherent growing cell lines into single cells using trypsin/EDTA
Seeding of 2000 single cells per well in ultra-low attachment 6-well plates
Use of serum-free MEBM medium (SFM) supplemented with 1xB27 supplement, 20 ng/ml human epidermal growth factor EGF, 10 ng/ml human basic fibroblast growth factor FGF, 20 IU/ml heparin, and 1% antibiotic/antimycotic solution
Incubation for 14 days

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