I have previously used CFSE for measuring the proliferation of lymphocytes. I now want to look at the proliferation of solid malignancy cancer cell lines, but I have read that CFSE may not be suitable because of uneven labeling of cancer cells. Can anyone recommend a good proliferation assay for cancer cells, preferably dye - FACs/plate reader based that doesn't involve metabolism of cells e.g. not MTT or Cell Titre Glo?
Well the easiest thing to do is to measure cell numbers over time, and make a growth curve. You can use Trypan blue exclusion to eliminate/count dead cells as well. Second, it depends on what you define as proliferation. Do you mean increased growth or decreased death? If you want to measure growth, I would recommend a BrdU/PI cell cycle analysis (example protocol: http://fccf.salk.edu/protocols/brdu.php). Briefly, BrdU is put into the cells and incorporated into replicating DNA over a specified time period. The cells are fixed and permeablized, and a BrdU-fluorescent antibody is used along with PI. Performing flow cytometry for PI vs BrdU, you can get a cell cycle analysis along with replication information. You can alternatively perform a Krishan PI cell cycle analysis if you just want cell cycle analysis without any info about BrdU incorporation. To rule out death, I would suggest a basic apoptosis assay using Annexin V/PI by flow cytometry, cleaved caspase 3, TUNEL, or all three to complement each other.
Hopefully this is helpful, good luck.
~Adam
Well the easiest thing to do is to measure cell numbers over time, and make a growth curve. You can use Trypan blue exclusion to eliminate/count dead cells as well. Second, it depends on what you define as proliferation. Do you mean increased growth or decreased death? If you want to measure growth, I would recommend a BrdU/PI cell cycle analysis (example protocol: http://fccf.salk.edu/protocols/brdu.php). Briefly, BrdU is put into the cells and incorporated into replicating DNA over a specified time period. The cells are fixed and permeablized, and a BrdU-fluorescent antibody is used along with PI. Performing flow cytometry for PI vs BrdU, you can get a cell cycle analysis along with replication information. You can alternatively perform a Krishan PI cell cycle analysis if you just want cell cycle analysis without any info about BrdU incorporation. To rule out death, I would suggest a basic apoptosis assay using Annexin V/PI by flow cytometry, cleaved caspase 3, TUNEL, or all three to complement each other.
Hopefully this is helpful, good luck.
~Adam
I wish it could help you to use this test : The Lymphocyte Proliferation Assay (LPA) is a test used to measure the ability of lymphocytes to proliferate in response to various stimuli such as candida, pokeweed and phytohaemagglutinin. These plant lectins are carbohydrate binding proteins that bind to cell surface receptors and activate cells in an antigen independent manner. Most people will respond to at least one of several common microbial antigens. LPA can be used to measure improvements in immunological function following antiretroviral therapy and to detect the presence of immune responses against specific opportunistic pathogens.
The 3H-thymidine incorporation assay which is used to determine the extent of cell division in response to a proliferation signal. 3H-thymidine which is a radioactive nucleoside and precursor of thymine found in DNA becomes incorporated in DNA strands of proliferating cells and can be measured to determine extent of cell proliferation.
To measure proliferation, the BrdU protocol is probably the best approach. However you have another protocols that you can use. Similar to MTT assay you have SRB, but is not based on the cell metabolism. You have the sulforhodamine B (SRB) assay that is used for cell density determination, based on the measurement of cellular protein content. You can see in this nature paper:
http://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.179.html
Hope this helps or give you another ideas!
As mentioned by others, cell counts with viability assay (e.g. PI exclusion you can do by flow - more precise than trypan blue & haemocytometer - you run thousands of cells in seconds) would be my choice providing there is a reasonable number of samples you need to assay. Great machine to do the counting for you is the old-fashioned Coulter Counter. This way you can identify relatively small differences you can later document by cell cycle analysis and BrdU incorporation. If you need to process large numbers of samples, don't expect very precise measurements, errors caused by handling small volumes of liquid are difficult to avoid. Going for the 96w plate-based assay, I would also suggest to fill the top and bottom rows and side columns with 300ul of water or PBS, cells grow there much faster than in the center of the plate and to have there liquid helps with evaporation issues over long incubation periods.
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