I want to design a Live- cell Calcium Imaging experiment on some cells. I have four groups Control, Stimulant, Stimulant+ Drug/ Inhibitor, Inhibitor Alone. Should I perform experiments for each experiment independently and then compare them or I can use combination like: Vehicle will act as baseline control for stimulant (recording in vehicle for 5- 10 min and rest 30 min after addition of stimulant); inhibitor alone as baseline control for stimulant plus inhibitor. But with this strategy I won't be able to compare inhibitor alone and control conditions... Also, the cells have to be treated with inhibitor for 90 minutes.... Please guide me how to design this experiment.
When I have done similar experiments in the past, I always did separate chamber slides for each condition, and recorded them separately. then I would compare all the results and plot them.
Yeah even I think that's the best methodology. Can you just detail me on how the specific condition of drug alone has to be recorded. In my case, the cells have to be pre- treated with the drug for 90 min. So should I Incubate the cells with drug for 90 min and when 5- 10 min are left for 90 min duration end, I will record the drug alone baseline and at the end of those 10 min or completion of 90 min, I will again add drug alone to record for rest 25- 30 min. Is this the right way to do this ?
you may need one more condition, where drug is applied at recording time, take measurements, then do another set where the cells were pre-treated. start recording with a new application of drug. the signal will be different, i would think. how will you load the pre-treated cells with Fura? doesnt that step take 20 minutes by itself?
While the cells are being pre- treated with the drug, at the end of 60 min, I will just withdraw 50 ul from the media the cells are in right now and mix the Calcium dye (eg 5ul) in that 50 ul. I will then add this mixture back to the rest of the media and gently swirl the dish. This will ensure the effect concentration of the drug to be maintained with the dye too being added to the cells.
be sure to keep the cells in the dark, once the dye is added, right. it can lose signal rapidly when exposed to ambient light.
Yeah Yeah... that will be surely taken care. So please tell me if I do this way, does the methodology that I mentioned above looks good for drug alone recording?
well, it looks ok, but it will be difficult to assess. I guess you could call it the "baseline" calcium signal following pre-treatment with drug. I would talk it over with some of your colleagues on hand.. make sure it sounds right to them as well.
You can use a 2-level, 3-factor factorial design for your experiment. You can test each combination of stimulant (yes, no), Drug (yes, no), Inhibitor (yes, no). This will give you 8 combinations. One of which will be a control. 3 of which will test each factor by itself. 3 of which will test a combination of 2 factors each and one run with all 3 factors. A design like this is far more powerful than the one you propose and can yield better information too. Its from a branch of statistics called Design of Experiments. This particular design is called a factorial design.
You already received excellent suggestions. One thought for the long duration treatment with inhibitors is to start the treatment during the loading and de-esterification of your calcium dye. Compare with one that you don't have the drug, if your loading and responses are the same, it would save a lot of time and you would get better cells at the end I think
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