I test the drug at 6, 12, 24 hours but find difficulty seed the cells at same day as 12 hours group will require me stay at lab 12 hrs or come very early next day. Can I do them as separate experiments? Like seed cells and add drug for 12 hour then do flow and add other cells and test after 24 hours? I compare cytotoxic drug vs normal .
Hi Heba
While testing the cytotoxic drugs or cytoprotective drugs, it is always recommended to plate the cells in same passage number, same cell density and same time for different testing intervals. For example, in this case you want to analyse the potential cytotoxic effects of a drug in a cell line. You should first ensure that the cells are in log phase of cell cycle. Then you should properly count and plate a requisite amount of cell population in your plate/flask. Be sure to leave the cells in this plate/flask for a sufficient period of time so that the cells may undergo atleast one division cycle. This will ensure that the cells you have plated do not experience any mechanical stress due to re-plating and pipetting.
When the cells have undergone one division cycle in this plate/flask, you may perform your experiment i.e. you can put the drug in the culture and take out a set of flask/plate at different intervals of 6, 12 or 24 h and perform desired techniques. Be sure to use a normal control everytime you are running a FACS or PCR analysis.
Good luck.
Like Sarisa said, You must to seed same density cells, because more cells mean difference in your results. Be careful with confluence, many cells in small space they will die and the experiments are affected.
Yes it is very important to have the same passage number and same plating density if you want to compare the different time points.
Hello Heba,
You have got very good suggestions above. I want to address your time management here which you are finding really difficult to deal with. Just seed your cells at day-1 in the morning, Give treatment next day at 8am (I think you can come at this time as it's not so early). Your 6 hrs will be completed at 2pm and 12 hrs at 8pm (you can stay for 9pm in lab). You can do FACS with the cells at the same day and put other cells in trizol and store for further RNA isolation and real time PCR. Next day, 8am in the morning, your 24 hrs. will be completed, you do FACS and then you can go for RNA isolation and of all three treatments group at same time and can perform PCR. Now u can also arrange 1-2 hrs up down as per your comfort...M sure that it will make your life easy.
Hope it helps !
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