Stage 1 of 3: Generation of Stable, Transfected Cell Lines: Kill Curve

If you are looking for practical information about how to generate stable, transfected cell lines, this first video of our three-part series describes how to determine the selection antibiotic concentration in a kill curve experiment. In this video, we use G418 Disulfate (Geneticin) in combination with CHO cells.
A link to the protocol can be found here:
www.toku-e.com/...
If you have any questions, please contact us: www.toku-e.com/...
For more information, visit our website: www.toku-e.com

[@mm669]

If you plate at 80% confluence, the cells will be over crowded by day two. How do you know if the cells are dying due to confluence or antibiotic? I know you have your control well without antibiotic to compare too, but that will be stressed too with the overcrowding. Wouldn't it be better to start with lower confluence?

[@toku-ecompany7075]

Thank you for your question. Whille it is true starting with a lower confluence will lower the risk of cell death due to overcrowding, this may also lower the transfection efficiency in the next step. Careful observation of the cells is key. If needed confluence can be adjusted based on the cell line, growth conditions, transfection method etc. You are welcome to contact '[e-mail]' if you have further questions.

[@iyanuoluwaniowolabi5658]

Hi. To prepare the selection media, I added the selection antibiotic to my normal culture media containing (90% DMEM, 10% FBS, 1%penicillin/streptomycin). Is this correct or should I have removed the 1%penicillin/streptomycin?

[@aysec7372]

For proper antibiotic concentration video shows as after 24 h check the cells and choose the lowest concentration that kills all cells within 10 days. How is it possible to decide after 24 h to 10th days result?
Should it be 10 days incubation and cell survival check?

[@toku-ecompany7075]

The video indicates you need to incubate the cells with different concentration of antibiotic for 24h and then check them. This is true but then you should keep the cells in culture for 10 days, check them regularly, refresh the medium with antibiotic and do the final evaluation after 10 days of culture. You are right that it ‘should be 10 days of incubation and then cell survival check’. The final evaluation is done after 10 days of culture with the antibiotic (with intermediate visual checks and medium refreshment every 3-4 days). Best, R&D Team.

[@aakifinayat8234]

can you please explain the dilution of parental cells? how did you diluted them?

[@toku-ecompany7075]

In order to prepare dilutions of parental cells, first the cells need to be dissociated from a culture vessel (using dissociation reagent e.g. trypsin) and resuspended in culture medium. Then the cells need to be counted in the suspension and an appropriate number of the cells (volume of the cell suspension) needs to be added to a conical tube with an appropriate amount of medium for each cell dilution in order to achieve the final cell concentration of 50 000, 100 000 and 200 000 cells/ml.
E.g. If the cell density of your starting suspension is 1 000 000 cells/ml, you will need to use the volumes specified below to have 5 ml of each cell dilution:
for a final cell conc (cells/ml) of 50,000 cells, you will use 0.25 ml of cell suspension and 4.75 ml of medium.
for a final cell conc (cells/ml) of 100,000 cells you will use 0.5 ml of cell suspension and 4.5 ml of medium
for a final cell conc. (cells/ml) of 200,000 cells you will use 1 ml of cell suspension and 4 ml of medium.
Hope this helps, and best of luck with you research.
Sincerely,
TOKU-E Team

[@ThanhNguyen-bs3mn]

Should we prepare selection media in large quantities (300 mL or so) once we know the optimal concentration of antibiotic or we should freshly prepare every time? How stable Hygromycin B in media storage at 37oC?

[@toku-ecompany7075]

We recommend preparation of a fresh selection medium just before adding it to the cells. Storage of culture media at 37°C is not recommended.

[@ThanhNguyen-bs3mn]

@@toku-ecompany7075 What would be a good way to quantify the kill curve of the dead cells? More quantitative way rather than just eyeballs them?

[@ThanhNguyen-bs3mn]

@@toku-ecompany7075 I was meant to put at 4oC

[@toku-ecompany7075]

In order to have quantitative results, you could use a fluorescent dye which stains nuclei of viable cells and measure the fluorescence of each well. You could also count the stained cells under the fluorescent microscope, however, it might be difficult to count all the cells in the wells. Alternatively you could do a viability assay based on cell metabolism (e.g. using MTT). However, be careful with cytostatic cells which could be underestimated by the assay and could still be present in the wells. This is why it is always good to check the wells visually. For additional questions, please email ‘[e-mail]’

[@toku-ecompany7075]

We have not tested Hygromycin B stability in culture media. We recommend adding the selection antibiotic to culture medium on the day of the treatment. For additional questions, please email ‘[e-mail]’.