I'm studying the proliferating effect of methylparabens on MCF7 by using Ki67 with flow cytometry & actually no results & I don't know why???
first I dissolved MP in ethanol(0.1M) & after subculturing in 6 well plate for 24 hrs, I put the parabens & incubate it for 7 days & then measure the proliferation with Ki67 with flow cytometry & I have used phenol red free media
I repeated this study several times with different doses & no results & I don't know where is my fault
Any help plz
You can try simple MTT, MTS or WST such as CCK-8 assays for examining the effects of your MP or any other drugs or compounds following cell proliferation kits from any supplier.
Good Luck!
you need to clarify further what do you mean by no result. do you mean negative flow cytometric staining? what do you compare against to judge the negative result?
if you are sure that your no result is negative staining, then ask whether you have done the staining procedure correctly. Protein like Ki67 is a nuclear marker which requires permebilization step and detergent like Triton X-100 or Tween20 included during staining.
You should measure proliferation over the course of time and not at an end-point. Its possible that there is a subtle difference early on while the paraben is still acting. Keep in mind that most reagents that you treat your cells will get metabolized and 7 days is a long time unless the effect is long lasting.
@Md. Farhadul Islam thanks for your reply, actually I have done MTT & there was no difference bet. MP & the control, there was no proliferation.
@ Win Sen Heng
I compare the proliferation of parabens in relation to control gp & estradiol as the proliferation of control was 84%, estradiol 95% while MP with different doses were less than the control & as regard the technique I used methanol for permeabilization, so what do you think where is my fault & thank you very much for your help
@ Shweta Tiwary thanks for your reply, yes every 2 days I change the media with the drug & as regard the effect of this substance it needs long time
I am always reluctant to use methanol for immunostaining, therefore I would recommend using paraformaldehyde for fixation and inclusion of either triton x-100 or tween20 for permeabilization step, stainings (primary and if you use secondary antibody) and washing after staining.
Marwa Kholief have you considered that there may not be a significant effect on proliferation under the conditions you are using. Sometime, 3D growth of a cell is very different than 2D growth. I will suggest trying a different assay such as colony formation in matrigel or soft agar. Also try including a cell line which you know shows a proliferation defect for sure ( I know that MCF7 does, but its not working for you) when treated with parabens, just to make sure that your technique is accurate.
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