Greeting everyone,
I have cancer cell line, and I need to measure cells proliferation. Does anyone have experience with this assay?
Thanks
shaima Jabbar
Cell proliferation can be determined in different ways: 1. The cell number increase with time and 2. increase in number of S-phase cells. The suggested protocols are one for each of them. Cell counting, I do not have anything to add but BrdU assay is little more technical. I found that Click IT EdU assay from Life sciences is easier than BrdU assay, you may want to research on it. Good luck.
PrestoBlue is quick, easy and cheap. You could also do XTT, which is suitable for long-term measurement and continued culture of cells.
Otherwise, normal cell counting is also sufficient.
Click-iT Edu would work. You can also use CFSE or Cell Trace Violet dyes from molecular probes and check proliferation using flow cytometry. These dyes can be longer in contrary to EdU/BrdU assays.
If you dont want to do flow cytometry and want quantitative data then best method is to use 3H-thymidine pulse and beta counter.
But all these methods will give you data comprising summation of Cell death and proliferation. Hence, it is very important to use a live cell dye before checking proliferation. You can use sytox-red/green dyes from molecular probes which would mark live cells and then you can analyse proliferation in these live cells and less contamination from dead cells. This assay can only be done using flow cytometer.
A good alternative is to use a PCNA Staining Kit (Life Technologies) to detect proliferating cells.
Cell counting with Trypan Blue solution is sufficient, but MTT, XTT or BrdU are more convinient and rapid for toxicological studies
You can use any automated cell counter or hemocytometer to count cell. Just make multiple wells with same seed concentration and remove and count cells from each well at a particular time. Should give you pretty good accuracy.
For cancer cell lines, i had used 2 ways for proliferation assay.
1) Non Radioactive MTS proliferation assay, is the best efficient and effective assay for cell proliferation but less accurate then radioactive assay. But very economical and much appropriate for used in first instance. Protocol is linked for your reference.
2) Radioactive: Its quite expensive and have to take lots of precautionary measure to perform this type of proliferation assay. I have used 3H Thymidine as radio-labelled indicator.
This above 2 methods are widely used, and measure the absorbance with appropriate instrument (mentioned in protocol)
http://www.promega.com/~/media/files/resources/protocols/technical%20bulletins/0/celltiter%2096%20aqueous%20one%20solution%20cell%20proliferation%20assay%20system%20protocol.pdf
If you stain your cells with propidium iodide and run them flow cytometer, you can determine the percentage of cells in various stages of cell cycle. This would be useful if you are studying cell cycle.
If you are dealing with suspension cells, you can always do cell count using haemocytometer, everyday for 4-5 days and determine the significance.
You can measure the cell proliferation by using Trypan blue assay and the proliferation curve can be measured by applying this equation= alive cells of the control - alive cells of the treatment /on alive cells of the control x100
MTT assay is a very cost-effective and simple way to compare cell viability. Also, you could do Western Blot to detect and compare levels of PCNA (Proliferating Cell Nuclear Antigen).
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