Comparison of measurement of surviving tumor cells by growth and clonogenic assays
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Objective To compare the effect of growth and clonogenic assays in measurement of surviving tumor cells after radiation. Methods The surviving cell number and surviving fraction (SF) of MCF 7 and Colo 205 cell lines following irradiation with 2,4,and 8 Gy were measured by using Courtenay Mills clonogenic and growth assays. Results In the clonogenic assay group, the detected surviving tumors had a clonic function. There was no difference of radiosensitivities between Colo 205 cells living in suspension and on adherence. In growth assay group, the relative surviving index reached the lowest level at the 6 th to 9 th days after culture and the mean value was close to that by the clonogenic assay. Conclusion Two methods reflect different aspects of cell proliferation. Growth assay can provide information on early proliferation kinetic events. The great merit of Courtenay Mills assay is that it claims to quantify not only surviving cells but cells with clonogenic potential, which is presumably a clinically relevant end point.Keywords:
Clonogenic assay
Cell counting
Suspension culture
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Clonogenic assay
Agarose
Spheroid
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Cell Survival
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Previous work has suggested that a radionuclide (3HTdR) uptake assay can provide a measure of drug and radiation sensitivity comparable with that given by the clonogenic assay. We have now extended this work to examine the radiation response of human small-cell lung cancer xenografts and cell lines with a wide range of plating efficiencies. Preliminary experiments using cultured cells indicated that irradiation of a single-cell suspension following disaggregation generally produced similar response data to those obtained when cultures were irradiated in situ and subsequently disaggregated. The response of eight cell lines to a single dose of 2 Gy was measured using both radionuclide uptake and clonogenic assays. There was no correlation between the two sets of data. Agreement between the radiation-response curves obtained using 3HTdR uptake and clonogenic assays was better for high plating efficiency xenografts (NCI-H69, COR-L51) than for low plating efficiency xenografts (COR-L24, COR-L31). Radionuclide uptake indicated very shallow response curves for the low plating efficiency lines. Altering the time of radionuclide addition from the standard Day 4 to other times between Day 2 and Day 6 did not greatly change the indications of radioresponsiveness provided by the assay. Growth-curve experiments for line COR-L24 showed that cell numbers at Day 4 after irradiation with 1.5 Gy or 3 Gy were very similar to those in unirradiated cultures. For NCI-H69, however, the cell numbers were very different for the different radiation doses. It appears that a high proportion of cells in lines such as COR-L24 take many days to show radiation damage as measured by reduced 3HTdR uptake.
Clonogenic assay
Plating efficiency
Radiation sensitivity
Radioresistance
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The human tumor clonogenic assay (HTCA) has potential value for studies of both the chemosensitivity and biology of human tumors. However, many technical problems including low plating efficiencies and the preparation of sufficient numbers of viable cells remain. In this study, an improved method for disaggregation of solid tumors increased the yield of single cells. Consequently, more than 10 anticancer drugs could be tested in 94 of 168 specimens (56%). Removal of peripheral blood lymphocytes from cell suspensions derived from effusions also improved colony formation. Adequate growth for sensitivity testing (>30 colonies/plate) was obtained in 122 cases (73%), inadequate growth for drug evaluation (5-29 colonies/plate) in 29 cases (17%), and no colony formation (<5 colonies/plate) in 17 cases (10%) of the 168 viable samples. The cloning efficiencies of cells derived from primary tumors (median 0.015%) were higher than those of cells derived from metastatic tumors (0.012%), and they varied with the location of the metastatic site. Clonig efficiencies varied markedly from specimen to specimen, and were unaffected by tumor histology, grade of differentiation, patient age, stage of disease, or prior chemotherapy. The HTCA is promising as a potential tool for studying the biology of tumors.
Clonogenic assay
Plating efficiency
Chemosensitivity assay
Metastatic carcinoma
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Abstract Radiation survival curves have been generated for 3 human tumor cell lines as a means of comparing and evaluating the validity of human tumor soft‐agar clonogenic assays. The assays investigated were the Hamburger‐Salmon, Courtenay‐Mills, Courtenay‐Mills plus additions, soft agar (no additions), and soft agar plus additions. The additions were formulated to supplement the media used in soft agar assays of primary ovarian and cervical carcinoma specimens. Supplementing the media with additions led to a 2‐ to 3‐fold increase in PE of CaSki cells but had no effect on the PEs of ME180 and OWI cells. Radiation survival curves were similar in all assays for CaSki and OWI but differed for ME180 cells. For ME180 cells, the Courtenay‐Mills and soft agar assays plus additions produced the most radioresistant curves (Do = 2.2 Gy); the cells were more responsive when assayed by the Hamburger‐Salmon method (Do = 1.5 Gy), and the soft agar and Courtenay‐Mills assays gave the most radiosensitive curves (Do = 1.2 Gy). These results demonstrate that the PE of human tumor cell lines may be increased with no effect on radiation survival; radiation survival may be altered without changes in PE and neither may be altered by applying modifications and supplements to existing clonogenic assays.
Clonogenic assay
Radioresistance
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The clonogenic assay is widely considered to be the most valid test for predicting tumor cell sensitivity to cytostatic drugs. In this study it was compared with early growth curves of human leukemic cell lines (HL-60, K562, Reh) after treatment with different types of cytostatic drugs (Adriamycin,cis-diamminedichloroplatinum(II):,1-beta-D-arabinofuranosyl- cytosine, and 5-fluorouracil) for 1 and 24 h. Following drug treatment two parallel cultures were started: a soft agar culture for the clonogenic assay; and a liquid suspension culture for vital cell counting by measuring esterase activity with fluorescein diacetate at different time points. The latter was recorded using flow cytometry during the following 3 days in 12-h intervals. For each drug concentration a survival factor was calculated from the growth curve between 24 and 72 h. This survival factor takes into account both the y intercept of the extrapolated growth curve and the slope of the growth curve. The dose-response curves resulting from either the survival factors or the clonogenic assay were always nearly identical. The results demonstrate that in established cell lines flow cytometric determination of vital cell increase rates provides a convenient alternative to the clonogenic assay for drug testing.
Clonogenic assay
Cell counting
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Clonogenic assay
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MTT assay
Clonogenic assay
Radiosensitivity
Chemosensitivity assay
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Clonogenic assay
Radiosensitivity
MTT assay
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Objective To evaluate the radiosensitivity of hepatocarcinoma cells HepG2 and BEL-7402 in vitro.Methods Using cell culture of human being hepatocarcinoma cell lines HepG2 and BEL-7402,we measured their surviving fraction by clonogenic assay and MTT after irradiated by 6MV X ray with doses ranging 0～10 Gy.Radiosensitivity index,D 0,was calculated.Results The D 0 of HepG2 was 1.5 Gy,SF 2(0.45±0.05).The D 0 of BEL-7402 was 1.6 Gy,SF 2(0.63±0.05).There was a good correlation between the results derived from MTT and clonogenic assay(γ=0.946, P 0.05).Conclusion The radiosensitivity of HepG2 is rather high compared with BEL-7402.MTT is a quick way to measure radiosensitivity of cells in vitro instead of clonogenic assay.
Radiosensitivity
Clonogenic assay
MTT assay
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