Karyomax giemsa stain solution

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.

Cells were treated with 0.1 µg/ml colcemid (GIBCO) for 3 h, trypsinized, washed twice with 1× PBS, and resuspended in hypotonic buffer (0.075 M KCl). After a 20 min incubation at 37°C, cells were centrifuged, and the pellet was gently resuspended in 3:1 methanol/glacial acetic acid fixative (vortex dropwise). Cells were washed with fixative three times, and a few drops were released onto an alcohol-cleaned slide and allowed to air-dry. Slides were counterstained with KaryoMAX Giemsa Stain Solution (GIBCO). Transmitted light images of metaphase spreads were captured using a 63× 1.4 NA lens, and the number of chromosomes per cell was counted using ImageJ software.

For assessment of cell viability, CH12 cells were either mock-treated (DMSO, Carl Roth) or incubated with 1 μM PARPi (Olaparib – AZD2281, Selleckchem) for 72 hr. Residual viability was expressed as percentage of cell viability of PARPi- over DMSO-treated cultures.
For genomic instability analysis, exponentially growing cells were treated with DMSO or 1 μM PARPi for 24 hr followed by 45 min incubation at 37°C with Colcemid (Roche). Metaphase preparation and aberration analysis were performed as follows. Cell pellets were resuspended in 0.075 M KCl at 37°C for 15 min to perform a hypotonic shock, and washed/fixed with 3:1 methanol (VWR)/glacial-acetic acid (Carl Roth) solution for 30 min at RT. Metaphase spreads were prepared by dropping fixed cells on humidified microscope slides, which were air-dried and placed at 42°C for 1 hr. Giemsa staining was performed by using KaryoMAX Giemsa Stain Solution and 1× Gurr Buffer (tablets, Gibco). Metaphases were acquired with the Automated Metaphase Finder System Metafer4 (MetaSystems).

Details regarding these experimental procedures have been described in our previous study [13 (link)]. Briefly, PC-3 cells were harvested and seeded in 12-well plate (100 cells per well). Cells were grown in the presence of γ-T3 at 2 μg/mL and/or Tie-2 inhibitor at 0.05, 0.1, or 0.2 μM. After 14 days, the colonies were stained with KaryoMAX^® Giemsa stain solution (Invitrogen), and the number of colonies formed was counted and normalised to that of the untreated control. Each experiment was repeated at least three times, and each data point represents the mean and standard deviation. Statistical difference was determined by a Student’s t-test and was considered significant if p <0.05.

Cell invasion was measured using the Matrigel® Matrix Chamber (Corning, USA). 30,000 LO₂ cells or 50,000 HepG2 cells were seeded on the matrigel layer together with medium containing 0.1% FBS. At the bottom of the transwell chamber, ten percent of FBS with medium was used as chemoattractant. The cells were then incubated for 96 hours. Cells on the upper surface of the chamber were removed using cotton swabs. On the other hand, the invaded cells were fixed using 3.7% formaldehyde, 100% methanol, KaryoMAX™ Giemsa Stain Solution (Invitrogen, USA) and Gurr solution for one minute each. Manual quantification of invaded cells based on four distinct fields were carried out (×40 magnification).