I am new to cell culture studies and workin g on colorectal cancer cell lines. I want to see the effect of a leptin on CRC cell proliferation and HCT 116 is used for that purpose (previously it was found that cell number was increased followed leptin treatment) . I have repeated my experiment several but do not see any significant difference in the cell number between control and treated group. I also added a positive control too and did find any significant change. this is extremely stressful as even with positive control I am not getting the result. I am using the following procedur:
1. Maintain HCT116 cells in DMEM (high glucose, L-glutamine, sodium pyruvate) with 10%FBS for 24 hours
2. Cells are starved in low serum media (0.1% FBS, high glucose with L-glutamine) for 24 hours
3. treatment is given to the cells in the same low serum media for 48 hours and then cell number is counted using BIO-rad automatic cell counter.
4. HCT 116 cells are from ATCC and i am using fresh new cells for my experiments.
what could be wrong I am doing? any Suggestions and ideas will be really helpfu.
You have more than one option here.
1- You have to look at the cells under the microscope to see if there are any dead cells, there may be something wrong with the counter.
2- For the positive control, it is very strange actually but you have to mention it and search for its effect on the HCT116 cell line in the literature
3- The concentrations you are using with the positive control is very low to cause cell death.
4- if you have to take care also for the confluency of cells in both the control and the cells to be treated, they should be the same before treatment.
Hope to hear about the positive control you are using
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