Clonogenic Assay: Adherent Cells

Hi, my name's Lum and I'm currently undertaking my PhD at the BE IDI Heart and Diabetes Institute under the supervision of Dr.Tom Carianne, who's head of the Epigenomic Medicine Lab. In this video, I'll be demonstrating the procedure known as Clon Genic Survival Assay. This is a 50-year-old technique, which enables the assessment of the impact of different treatments such as exposure to radiation or chemical agents on cellular reproductive viability.

Congenic survival assay is a central technique in radiation biology for evaluating the radiation sensitivity of different cell lines and testing the efficacy of different radio protective or radio sensitizing compounds. When using an A adherent cell line, the cryogenic survival assay consists of three phases. First, the treatment of a mono layer of a adherent cells.

Second, the preparation of a single cell suspension and plating of known number of cells and painted dishes, and third, staining, counting, and colonies formed after a period of incubation. In this video, I'll be using immortalized, human cino site cells to demonstrate how polygenic Bible assay can be used to determine the radio protect properties of the natural antioxidant. 1225 M flas are seeded of cells in five mils of media beforehand, so that on the day of the experiment there are 1 million cells per flask.

50 microliters of the drug is added at different concentrations to the five mils of media in each appropriately labeled flask to achieve the required concentration, five concentrations are used as well as one vehicle control in this case, PBS. After adding the drug, the cells are incubated for one hour in the conditions appropriate for the cell line used After incubation. The flux, excluding the control group, are irradiated at the chosen dosage, which in this case is four gray produced by a gamma ray emitting cesium source.

After irradiation, the medias poured off and each flask is washed with two mils of PBS for five minutes. After pouring off the PBS two mils of 0.05%trypsin are added to each flask to detach the A adherent cells from the flask. Bottom five milliliters of DMEM containing 10%fetal bovine serum is then added to each flask to deactivate the trypsin.

The full contents of the fasc are then transferred to labeled 10 milliliter falcon tubes. Once complete, these tubes are centrifuge for five minutes, after which the supinator is discarded carefully without disturbing the palate, and five mils of crecy media is added to each tube. The concentration of cells for each sample is ENC calculated as well as the volume needed of each sample to achieve final concentration of cells to be plated out into five Petri dishes per sample with five milliliters of media each.

For example, for the control group, 100 cells are plated per dish, so the volume needed to achieve 500 cells per 25 milliliters is calculated for more concentrated samples, perform either a 10 times dilution or a 100 times serial dilution to minimize the error involved when using volumes less than 100 microliters. Now the volume previously calculated is prepared into a Falcon tube containing 25 milliliters of media for each sample. The samples can now be transferred to previously labeled pre to dishes, adding five milliliters to each dish for easier handling and transport.

Store the dishes in a large, sterile, or disinfected container. The dishes are then placed in a humidified incubator in the appropriate conditions and incubated for eight days. If using keratinocytes after eight days, carefully remove the dishes from the incubator and lay them all out one by one inside a fume hood.

Be careful to handle the place gently so that any colonies that have formed aren't disturbed. Remove the lids and add five mls of 0.9%saline to each dish to wash them. Aim the prepare tip into the corner of the dish and avoid touching the bottom of the dish so that any colonies are not accidentally scraped off.

After five minutes, carefully aspirate out the saline. Now, colonies are fixed with three milliliters of 4%formula, and the leads must be replaced to avoid evaporation. After 15 minutes to half an hour, the formula is aspirated out and disposed of appropriately.

The colonies are now ready to be stained with 0.1%Crystal violet, five milliliters are added to each dish and the lids are replaced after half an hour to one hour. The dye is then washed off with distilled water and the dishes are inverted and allowed to dry overnight. The colonies are now ready to be counted.

Colonies can be counted manually using a dissecting microscope. Any cluster of cells containing 50 or more cells is considered to be a colony. And that concludes our demonstration of the Congenic survival assay.

The main advantage of this assay over other techniques is that it's incredibly sensitive and can detect survival over a large range even after six logs of cell kill. This, this sensitivity is achieved by plating larger cell numbers. This essay can easily be used for other purposes, such as test, such as testing cytotoxicity.

Thanks for watching.