Cytoselect 96 well cell transformation assay kit

The colony formation of the cells was conducted using CytoSelect 96-well cell transformation assay kit (Cell Biolabs, San Diego, CA) according to the manufacturer’s instructions. In brief, cell suspension (7.5 × 10³) was prepared in 0.4% agar with DMEM and seeded in 96 well plates pre-coated with 0.6% agar solution. The treatment was added to each well after solidification of the cell agar. The cells were lysed after seven days of incubation. The DNA were stained with CyQuant GR dye and determined by Victor X4 fluorescent plate reader (PerkinElmer, CT, USA) using a 485/520 nm filter.

Anchorage-independent growth of cells was determined using the CytoSelect 96-well Cell Transformation Assay kit (Cell Biolabs, San Diego, CA, USA) by performing soft agar colony formation assays. The numbers and morphologies of colonies were determined using an inverted phase-contrast microscope (Olympus, Tokyo, Japan) after 10 days of seeding. To quantify the anchorage-independent growth, colonies were lysed with lysis buffer and CyQuant GR dye was added to generate fluorescence. Finally, fluorescence was measured using a fluorometer with filter setting at 485/520 (Wallac Victor3 1420 mutilabel counter, Perkin Elmer, Waltham, MA, USA). Data are presented as the means ± SD of 3 independent wells.

Soft agar assay was achieved with CytoSelect 96-Well Cell Transformation Assay kit from Cell Bio labs, Inc as described in the manufacturer's protocol. The total number of transformed cells/well was calculated from the cells/milliliter in soft agar as given in the manual.

The soft agar colony formation assay was performed using the CytoSelect 96-well cell transformation assay kit (Cell Biolabs, San Diego, CA, USA). Human cervical carcinoma cell line HeLa cells (JCRB Cell Bank, NIBIOHN, Osaka, Japan) were used as a positive control since they are known to form colonies in soft agar medium (Kusakawa et al.^{11 (link)}; Ke et al.^{12 (link)}; Seo et al.^{13 (link)}). The cells were maintained in Eagle’s minimum essential medium (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 0.1 mM non-essential amino acids (Thermo Fisher Scientific). The primary human cardiomyocytes (HCM) (PromoCell, Heidelberg, Germany) were were maintained in Myocyte Growth Medium (PromoCell). HeLa cells (1%, 100 cells; 0.5%, 50 cells; 0.1%, 10 cells; and 0.01%, 1 cell) were spiked into 1.0 × 10⁴ HCM and grown in soft agar for 10 or 20 days. The cells were lysed and absorbance was recorded on a microplate reader (DS Pharma Biomedical, Osaka, Japan) at a wavelength of 485/520 nm filter set. The experiment was performed in triplicate.

Anchorage-independent transformation assays were performed using a CytoSelect 96-well cell transformation assay kit (Cell Biolabs, Inc.). Briefly, equal number of cells were plated in soft agar in a 96-well plate and cultured in the presence or absence of indicated inhibitors. The transformation was determined according to the protocol provided by the manufacturer.