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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 'info.eu@toku-e.com' if you have further questions.

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

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.

Thank you for question. Yes, we keep the cells in selection medium until we have enough cells for freezing (to ensure high purity of cell line stock). Later on the cells can be cultured without the selection antibiotic, however, they should be monitored for expression of gene of interest. If the percentage of positive cells drops during the selection-free culture, the selection antibiotic is re-introduced.

Conditioned selection medium is prepared by adding the following components to the fresh culture medium: - The conditioned medium - the medium which was collected from the cultured cells - to achieve the concentration of 10 % (so e.g. 10 ml of the conditioned medium added to 90 ml of the fresh medium) - The selection antibiotic to reach the concentration determined in a kill curve experiment - explained in the video ‘Generation of Stable, Transfected Cell Lines: Kill Curve (Stage 1 of 3)’.

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.

@@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?

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

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 ‘info@toku-e.com’

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 ‘info@toku-e.com’.