GGS LIVE - Clonogenic Survival Assay

Yo Nerds,


Method: Clonogenic Survival Assay (CSA).

About: Clonogenic survival assay is a long term cell viability assay in which ability of a single cell to form a colony (proliferation capacity) is scored. 

What: Clonogenic survival assay after DNA damage induced by Drug X using two different cell lines: wild-type and DNA repair defficient mutant.

Clonogenic survival assay is performed differently for suspension and adherent cells. As I am working with suspension cell at the moment, I will explain only how to perform CSA for suspension cells.

Around 5 x 10^5 cells is enough to perform clonogenic survival assay with four different doses of Drug X.
If we perform clonogenic survival assay for the first time using a mutant cell line (or using a particular drug for the first time) it is important to plate different number of cells for each dose as it is hard to predict sensitivity, for example:

It is also important to seed cells in triplicate. This is because we want to base our result on a three different numbers what makes it more accurate (not on a single number but on avarage of three). When we have our cells and numbers ready we have to prepare plates. Different plates can be used for this purpose but in our lab we are using the triple vent petri dishes (see on the picture further on).

Special media has to be used in the case of suspension cells, in our lab we are using a methylcellulose based media. Methylcellulose is added to the media as a thickener so our "mobile" suspension cells do not move after we plate them. This prevents a single cell to give rise to multiple colonies. You can imagine that if media is not thick colony could split resulting in extra number of colonies at the end of the experiment. Once the media is placed in dishes we can start plating cells. For that purpose a dilution method is used, as show on the picture below:

We prepare our cells so we have a 10^5 cells per ml and we place them in 24 well plate (picture above).
We treat this cells with Drug X at doses 0, 2, 4 and 8 mg/ml (in the 0 dose we treat cells with the solvent used to dissolve the Drug X only). We incubate the cells at 37 degrees Celsius for specific time and after that time we dilute cells twice each time by factor of 10 (as on the picture above). This way we obtain three cell solutions in which we have 100 000, 10 000 and 1000 cells in total per ml. This means that if we use 100ul from each solution we will plate 10 000, 1000 and 100 cells onto dish, respectively. After the cells are plated we incubate them for 7-14 days depending on how fast cells grow and give visible colonies (see picture below).

Picture taken by Kliszczak M.

Three representative colonies are indicated with red arrowheads. We count colonies and then we calculate the survival according to the table shown below:

After the calculations we plot the % of relative survival against the doses of Drug X and we obtain a plot like on the picture below:

From this experiment we can conclude that mutant cell line is more sensitive to Drug X than wild-type cells and that this sensitivity is dose dependent. We have to repeat this experiment at least three times to obtain reliable result. Remember that all experimental steps (cell number, drug doses, time of cell treatment) has to be optimized and each time performed in the same way to minimize the errors.

I hope u enjoyed it:)

Maciek GGSTEAM