Effect of different doses of X-ray irradiation on survival of human esophageal cells
Robert Hofstra, Noriyuki Koibuchi, Suthat Fucharoen in Advances in Biomolecular Medicine, 2017
Clonogenic assay was performed to assess the survival of both cells. Cells were cultured in a 10 cm culture dish at a density of 1 × 106 cells/10 ml medium. After the cells had reached 80% confluence, CHEK-1 cells and TE-8 cells were irradiated with 2 Gy, 4 Gy, 6 Gy and 8 Gy doses of X-ray irradiation. Immediately following irradiation, the cells (500 cells/4 ml medium) were seeded in 25 cm2 Falcon tissue culture flasks. Following 14 days of culture at 37°C and 5% CO2, the cells were washed with PBS (Phosphate Buffer Saline), then fixed with 99.5% ethanol, and stained with 0.5% crystal violet in H2O: methanol (1:1) for 30 min at room temperature. The cells were then washed with tap water and air-dried. The total number of colonies containing >50 cells was counted using a binocular light microscope (Olympus Corporation, Tokyo, Japan). The Plating Efficiency (PE) and Survival Fraction (SF) were calculated using the following equations (Buch et al., 2012, Franken et al., 2006):
Structure-Function Elucidation of Flavonoids by Modern Technologies
Dilip Ghosh, Pulok K. Mukherjee in Natural Medicines, 2019
Another cell-based assay frequently used for evaluating the anticancer activity of any drug is the clonogenic assay. This is mainly used to evaluate the loss of reproductive integrity or death of the cells after treatment of the same with any radiation or any chemotherapeutic agents. The basic principle of this assay lies in the fact that the ability of a single cell to grow into a large colony can be visualized by the naked eye/microscopically. This gives a clear cut implication that these cells have retained their reproductive integrity. Loss of this ability as a function of radiation and chemotherapy can be deduced by the dose-survival curve. Although the assay produces accurate results, the only drawback is that the process is quite time consuming. Eventually, the percentage of cells that survive the drug treatment is measured and analysed further. Normally in this type of assay, limited numbers of cells are seeded initially and on completion of the incubation, the cells are fixed and stained to draw a conclusion. As far as the functional activity of the flavonoids are concerned, different dosages of the flavonoids can be used to determine the dose-survival curve and related cytotoxicity. The detailed protocol for this assay has been described elsewhere (Munshi et al. 2005).
Detection of Metastatic Tumor Cells in Bone Marrow
Adrian P. Gee in BONE MARROW PROCESSING and PURGING, 2020
Another method for detection of minimal marrow disease is the use of the clonogenic assay. This technique, first reported by Hamburger and Salmon,30 was an attempt to define poor prognostic malignancies by analyzing their potential for colony growth in vitro. Several investigators have used a modification of this method to grow neoplastic colonies from bone marrow.17,31–34 This has been proven to be superior to routine histologic analysis. However, the number of tumor cells capable of producing colonies in this assay (plating efficiency) is low, even if highly malignant cells are used (e.g., neoplastic cell lines).35,36 Consequently, the sensitivity of this method is low, and studies have shown that immunologic detection techniques are superior. Despite this drawback, the clonogenic assay is useful for malignancies where monoclonal antibodies that can differentiate tumor cells from normal hematopoietic cells are unavailable (e.g., in Hodgkin’s and non-Hodgkin’s lymphoma).32
Increased uptake of doxorubicin by cells undergoing heat stress does not explain its synergistic cytotoxicity with hyperthermia
Published in International Journal of Hyperthermia, 2019
Anirudh Sharma, Sanem Özayral, Julia S. Caserto, Rosemarie ten Cate, Nicole M. Anders, James D. Barnett, Sri Kamal Kandala, Elizabeth Henderson, Jacqueline Stewart, Eleni Liapi, Michelle A. Rudek, Nicolaas A.P. Franken, Arlene L. Oei, Preethi Korangath, Fred Bunz, Robert Ivkov
For all Dox + HT combination experiments, Dox concentrations and HT thermal doses were chosen as approximately, the IC50 condition (Figure 2(a) and Table S1). For Dox + HT (simultaneous) experiments in HCT116, for example, 5 ml of pre-warmed (37 °C) media containing Dox at 1 μg/ml concentration was added to the 5 ml of existing media in the T25 flask, so that the final concentration of Dox in the flask was 0.5 μg/ml. Immediately following Dox addition, the flasks were immersed into the water bath with the surrogate flask containing 10 ml of media and treated for 60 min at steady-state temperature of 42 °C ± 0.3 °C. This process of adding the Dox to the flask and immersing in the water bath was ∼1 min. The 90-min exposure to Dox began when Dox was added to the flask. Total exposure time to Dox + HT, including, warm-up, cool-down and sample processing was 90 min. Following exposure, cells were prepared for clonogenic assay as described below.
3D high resolution clonogenic survival measurement of xrs-5 cells in low-dose region of carbon ion plans
Published in International Journal of Radiation Biology, 2023
Dea Kartini, Olga Sokol, Chutima Talabnin, Chinorat Kobdaj, Marco Durante, Michael Krämer, Martina Fuss
For recovering cells from matrigel, the medium layer was discarded and 22 µL of dispase (Corning) was added and incubated for 1 h to dissolve the matrigel matrix. The dissolved gel was collected into microtubes which were filled up with medium until the total volume of suspension reached 1 mL and then centrifuged for 5 min (2500 rpm, 4µL of trypsin was mixed with the cell pellet and incubated for 3 min to separate cell clumps into single cells. Afterwards, 900 µL of medium was added to deactivate the trypsin. Cells were counted using the Beckman Coulter counter using a profile with 1:50 dilution and 7 − 18 µm particle size. The average number of cells obtained was 75,048 cells per well with an uncertainty of ±11.5%. We find the cell number obtained is sufficient for performing the clonogenic assay.
Radiosensitizing Effect of Curcumin on Human Bladder Cancer Cell Lines: Impact on DNA Repair Mechanisms
Published in Nutrition and Cancer, 2022
Joyce Azzi, Anthony Waked, Jolie Bou-Gharios, Joelle Al Choboq, Fady Geara, Larry Bodgi, Mira Maalouf
After assessing the cellular viability, we performed the clonogenic assay to confirm the previous results. A significant decrease in cell survival was observed in UM-UC5 cells upon treatment with Curc (p < 0.01). The percentage of the surviving fraction (SF) was 74.94% ± 11.16% and 62.93% ± 3.33% in UM-UC5 after treatment with 5 and 10 µM Curc, respectively (p < 0.01, Figure 9A). Treatment with 2 Gy X-rays resulted in a SF value (SF2) of 27.97% ± 3.26%, but no additional effect was observed when cells were previously treated with Curc (p > 0.05, Figure 9A). Regarding the UM-UC6 cell line, the cell survival was significantly inhibited by 46.45% ± 4.31% and 12.62% ± 2.37% when cells were treated with 5 and 10 µM Curc, respectively (p < 0.01, Figure 9B). UM-UC6 was the most radioresistant cell line with a SF2 value of 39.93% ± 4.70%. This SF value was highly decreased to 5.36% ± 0.88% after the combination treatment (IR + 5 µM Curc) (p < 0.01, Figure 9B).