Hi, everyone
I am a novice in biology, I am now conducting experiments on MDA-MB-231 cell lines. my results from cell viability assays are not like my expectation, I ,therefore, hope experts in this field could help me out. I will list some questions below.
1. when I did cell counting, I often dilute to 16-20 cells per square, so I wonder that the number of the cells is enough to be a representative sample for the whole population. because if the number of cells is higher, I find it difficult to count them.
2. I seeded 10^4 cells/well in a 96-well plate, and my positive control has a quite low signal in comparison to the previous results that I took from my senior. do you have any recommendation please tell me.
3. I carry out cell viability by Ws-1 assay. I read its protocol and it tells that after seeding cells, we will wait for it in 24 hours. I think the aim of this step is to await them to adhere to the well surface, so could I reduce the time or not.
Thank all of you for spending your time to read my question, I hope to receive your answers as soon as possible.
1. I'm not sure that I've correctly understood what you're saying here, but... You shouldn't be counting only one square (assuming that you are referring to a haemocytometer), but rather the four corners and the middle - this is what makes your count relatively representative of the population. You should be able to count with more cells than that in a square (even thinking specifically of MDA-MB-231) so long as they are not overlapping too much and even then clumps can normally be identified as a # of cells. If you're getting that problem then you probably just need to mix your cells better.
2. As in, compared to results achieved by a previous investigator? Was it carried out in an identical manner? Cell density, cell line, everything? Also, positive control for what exactly? It may be the assay not the cells. Also, exactly how different is your result compared to the previous?
3. I haven't carried out that assay, but yes your cells need to adhere, unless you don't want them to for some reason. In my experience, 231s can adhere far quicker than that, but it does no harm to be sure. Why don't you just test their adherence at different time points?
1. Yes, I used haemocytometer, and I mean the number of cells in a small square is 16-20 and I count 4 squares and took the average. could you recommend how many cells are proper to get a relatively representative sample.
2. I did experiments on the same cell density, cell line. the positive control for inhibiting cell proliferation. the results of a previous investigator is two-fold higher than mine. the previous result reached 30% inhibition, but my result only reached 19-24 percent.
3. I will test it at different time points.
thank you for your help a lot
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