Question

I have to develop my own experimental procedure for my lab covering cell culture. We’re using HEK932T cells and I wanted to test the affect salt has on the cells. I know how to culture cells but I’m not sure how to test the cells with the salt. We’re using a 12 well plate and each well is 1 mL. I know how to count the cells but I don’t know how many microliters to add to each well. How do I determine how many microliters of the cells to put into each well? When I add the salt do I dissolve it in water and then just add it straight to the cells in the wells? Once the salt is added then how do I observe the cells? Can I just put the whole plate under an inverted phase contrast microscope or do I have to add dye and view it under a compound microscope? Also what control should I have? I don’t know if I should just have the cells in the well as my control or add water to the cells for my control?

Answer 1

To test the effect of salt on the cells, the steps would be as mentioned below:

1. Decide the concentration of the salt you desire to test and make a higher concentrated salt solution in serum-free media so that in one ml of culture media in the plate, you end up adding salt concentrate with volumes lower than 10µl. Your final test concentration should be achieved in one ml media.
2. When you are passaging the cells from your parent culture plate/ flask to the 12 well test plates, after centrifugation of cells from the parent plate/flask resuspend the cells in 1200µl of media and count the cells as per standard procedure using a hemocytometer.
3. Prepare the 12 well plate with 900µl media and add 100µl of resuspended cells to each well, so that now the number of cells in each well is approximately the same and you can deduce the count /well.
4. Once the cells adhere to the plate, before starting the experiment, we remove the old media and add 1ml of fresh media in each well which has cells adhered to the bottom.
5. Mark the top 6 wells as controls and the bottom 6 wells as salt treatment.
6. Add 10µl of only serum-free media in the control wells and add 10µl of concentrated salt solution in the salt treatment wells.
7. Let's assume your incubation time is 3 hours. After the end of three hours, you can directly observe the cells under an inverted phase-contrast microscope to see the effect of salt on the cell morphology in the salt-treated compared to controls.