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.

When we want to actually see what happens with a "X" drug on a "Y" cell line, we are just looking what happens to the treated cells as compared to the control ones as explained below and illustrated in the attached documents.

In order to have a more than rough idea about compound-of-interest-induced in vitro anticancer effects, we routinely perform computer-assisted phase-contrast microscope analyses of living cells during 48 to 72h of observation. The cells (whatever normal or cancerous) are cultured in plastic flasks containing buffered media, but in an incubator without CO2 regulation (while thermoregulated at 37°C). The media we use are described in the attached articles (see below).

You can see in the movies here attached the “morphological effects” observed with two very distinct compounds. Each film results from 1,080 digitized images taken every 4 min during 72h and compressed into a 40-60- sec-film. Each film can be seen with softwares freely available from the net. The counter with yellow numbers that you can see in some of the attached films gives you an estimation of the “dynamics” (in terms of number of hours of observation) of the cell behavior.

The attached films illustrate two markedly distinct situations.

The first set of experimental condition refers to human HS683 malignant oligodendroglioma cells (see QVM-1 Le Mercier et al., Neoplasia 2009) left untreated (control; QVM-2 Film Hs683 Control) or treated (QVM-3 Film Hs683-Ferrocifene) with a ferrocifene derivative. These are these morphological observations relating mainly to the enlargement of the treated Hs683 cells as compared to the control ones (without “frank” death processes (see below what we mean as “frank death processes for sphaeropsidine A)) that led us to formulate the hypothesis about potential induction of senescence. The rest of the story is detailed in QVM-4 Bruyere-Mathieu et al. J Inorg Biochem 2014). We estimate that we are facing here CYTOSTATIC effects.

The second set of experimental conditions refers to human SKMEL-28 melanoma cells left untreated (QVM-5 SKMEL-28 Control) or treated with sphaeropsidin A (QVM-6 SKMEL-28 Sphaeropsidine A). These experiments actually helped us to appreciate the CYTOTOXIC effects of sphaeropsidin A (a more “in-depth” analysis is provided in QVM-7 Mathieu et al. CMLS 2015). The attached document labeled QVM-8 Van Goietsenoven et al. FASEB J 2010 provides a view about various sensitivity levels of melanoma cell lines to pro-apoptotic cytotoxic insults.

The “morphological approach” that we routinely employ can also be used to see how develop primary cultures from a human cancer biopsy (here a GBM biopsy, in the left-handed bottom part of the film "biopsy" – QVM-9 GBM Biopsy).

Whatever it can be, once a researcher “saw” what happens in the flask containing the cancer cells (when using her / his compound(s) of interest), she / he can thus use other technique, including flow cytometry, to analyze precise cell cycle kinetics or cell death-related parameters (described by Galluzzi et al. 2012).

Best regards

Robert