I am new in the radiotherapy field and I need to set some experiments to determine the sensitivity of several cells lines to radiation.
I read a lot of papers and they look like to achieve my goal They use a technique called survival fraction assay, which is simply a clonogenic assay with some modifications.
My questions are:
How long take a cell line to die after radiation?
Why I can not use, for example MTT assays to check viability 72 h after radiation (different doses) and determine the IC50?
I am seeding cells and irradiating them with different doses and then doing MTT 72 h later. But my cells even at the highest doses of radiation, looks like controls. Do you know why??? They need more time to died o what?
I appreciate your helps in this matter.
Best
After 72 h cells are going to die due to unavailability of space in 96 well plate because only 4000/cells are survive in one well of 96 well plate. Increase your radiations intensity then check their results
Dear Ali,
First of all, IC50 (referring to the concentration) is not an appropriate term when using radiation.
Second, number of initial cells, type of cells, dose and time of radiation, possibly distance to the radiation source, presence/lack of antioxidants in the culture media, are all determinants. Besides, don't remember that the cells should be completely exposed to the radiation. Actually, I guess that plate lid may absorb some rays. Finally, you can investigate the ROS production in treated/untreated cells as an additional study. Good luck
Hello Ali Calderon-Aparicio
Aberrant cell proliferation rates and the propensity of individual cells to survive and propagate over long periods of time are the two hallmarks of cancer. You may use cell proliferation assays to measure the former, while the colony formation assay is to used to assess the latter. Both proliferation and colony formation assays play a key role in cancer research and rely on accurate determination of cell number within a culture, however they differ substantially in a few important aspects.
Cell proliferation assays quantify the rate in which a population of cells increase in number over time after initial cell seeding in a microplate. Endpoint cell proliferation assays provide a single snapshot in time of cell number after a predefined experiment duration that is compared across treatment conditions.
On the other hand, clonogenic assays are a little different. Clonogenic assays measure the ability of cells to retain their reproductive integrity over a prolonged period of time. This is an important feature as it reveals phenotypic effects that require time and possibly several cell divisions to develop. When one is analyzing therapeutic resistance this becomes particularly important. You cannot identify drug resistance using short-term cytotoxicity assays.
So, clonogenic assays are widely used in the field of cancer research as the formation of clones is interpreted as a trait of cancer cells with tumor-initiating capabilities. While this assay was initially used in the field of radiobiology, it has now become a standard tool in cancer research to evaluate cellular growth.
I agree with what Alireza Naderi Sohi has mentioned namely, IC50 (referring to the concentration) is not an appropriate term when using radiation. Do not mix up cytotoxicity assay (MTT) with clonogenic assay. These are assays used for two different purposes. Proliferation assay and the colony formation assay provide valuable information about the characteristics of transformed cells. While often one or the other assay type is selected for inclusion in a given study, together they will provide complementary data and greater overall insight into cancer cell behavior.
Clonogenic assay is the gold standard method for measuring the radiosensitivity of cancer cells. So, to determine the sensitivity of several cell lines to radiation you will need to perform clonogenic assay and not MTT assay.
Regards
Malcolm
Further to Malcom Nobre's answer:
You want to measure the survival curve of your cells so as to determine the sensitivity of your cells/cell line to ionizing radiation(s). The survival to measure is that of the cells' reproductive integrity. To do this you prepare a sufficient number of flasks or Petri dishes with equal numbers of cells (e.g., 200 cells per T25 flask) and after they have attached, irradiate them with the desired doses to cover a range of surviving fractions(SFs) from 100% (control) down to 0.1% or 0.01%. Thus, about 5 flasks (10^3 cells total between them) for the low doses and increasing numbers of flasks per dose to obtain comparable numbers of clonogenic survivors for the flaks at each dose. You do not want to increase the number of cells per flask to get the same number of survivors for each dose since the higher density of "dead" cells per flask will alter the response of the cells.
Generally, the curve shape, when plotted as Ln(SF) vs Dose, will be an initial exponential, followed by a "shoulder", and then a terminal exponential. The equation fitting this curve will likely be SF = exp(-D0init)[1-exp(-D0final)]^n, where D0init is the initial reciprocal slope of the initial linear region, D0final is the reciprocal slope of the terminal linear region, and n is the extrapolation number (obtained by extrapolating the terminal slope as a straight line (on the semilog plot) to zero dose). These 3 parameters define the cells' sensitivity to the radiation used.
The loss of reproductive integrity is a modified exponential (hence the linear regions on the semilog plot), hence no dose kills all of the cells. You merely reduce the survival probability low enough that for the number of cells exposed, it is
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