Colony Formation Assay for Adherent Cells

The procedures for the colony assay of adherent cells were performed as described in previous articles [24] (link)–[27] (link). In brief, both sham and exposed cells were seeded in 10 cm tissue culture dishes (Orange Scientific, Braine-l'Alleud, Belgium), 500 cells per dish, containing 15 ml of DMEM. Cells were allowed to attach to the Petri dishes for 12 h before ELF-EMF exposure and the attachment of cells was verified using a microscope. After 144 h of ELF-EMF exposure at 37°C, all the exposed cells were transferred from the exposure area to the magnetic shielded mu-metal box in the same incubator for 8 d. During clonal expansion, both the sham and exposed cells were not disturbed until the day for harvesting. The colonies of each petri dish were washed with PBS and fixed with 3 ml of Carnoy’s solution (methanol:acetic acid 3∶1, v/v) for 3 min. Then cells were washed with PBS and fixed in 100% methanol for 30 min followed by staining with KaryoMAX Giemsa Stain Solution (Gibco BRL, Grand Island, NY) for 10 min. Each dish was washed with water and dried overnight at room temperature. A colony is defined as a cluster of more than 50 cells. To perform the statistical significance tests we conducted colony formation assay in triplicates.