Isovolt titan x ray generator

Cells were seeded in 60 mm Petri dishes (Thermo Fisher Scientific, Roskilde, Denmark) at a density of 720 to 7200 cells per dish and incubated for 24 h at 37 °C before the addition of the KIs CC115 (1 μmol/L), AZD7648 (5 μmol/L), or Sapanisertib (0.5 μmol/L). We delivered 2 Gy IR 3 h later by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany). After 24 h of incubation, the medium containing the drug was replaced with fresh medium. Cultures were incubated for 1–3 weeks until colonies were formed. Colonies were stained with methylene blue (#66725, Sigma Aldrich, Munich, Germany) for 30 min at room temperature. Clusters containing 50 or more cells were considered to be a colony. Image analysis software (Biogas Kobi 1.2) was used to count the colonies (Biomas, Erlangen, Germany). The determination of plating efficiency (PE) involved calculating the ratio of the colonies formed to the initially seeded cells. The survival fraction (SF) was then computed by dividing the number of colonies formed by the PE and multiplying the result by the number of cells initially seeded [59 (link)].

The acute monocytic leukemia cell line THP1 (ATCC^® TIB–202™), promyelocytic leukemia cell line HL60 (ATCC^® CCL–240™), and the multiple myeloma cell line RPMI8226 (ATCC^® CCL–155™) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). All the investigated cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (10 U/mL), and streptomycin (50 μg/mL) in standard conditions: 37 °C, 100% humidity, and an atmosphere of 5% CO₂ and 95% air. Cell viability was systematically controlled using 0.4% trypan blue. In all experiments, cells in a logarithmic growth phase were used when their viability was above 95%.
One concentration of the drugs for each cell line was chosen for the study: THP1: 0.3 µM; HL60: 0.7 µM; RPMI8226: 3 µM. These were the same as in previous studies evaluating the biological properties of melphalan analogs [14 (link),15 (link)]. In all experiments, cells were treated for 4 h, 24 h, and 48 h. Simultaneously, control cultures were incubated similarly without further treatment. For the immunostaining assay, a positive control from irradiated cells was prepared. The cells were irradiated with 1 Gy of ionizing radiation by an ISOVOLT Titan X-ray generator (GE, Ahrensburg, Germany) [51 (link),52 (link)].