You should be much more precise in your question.

It seems to me that you label "cell proliferation" and "cell growth" as synonyms, which is not the case.

What do you want to do exactly?

Best regards

Robert

You should be much more precise in your question.

It seems to me that you label "cell proliferation" and "cell growth" as synonyms, which is not the case.

What do you want to do exactly?

Best regards

Robert

Trypan blue is a vital stain used to selectively colour dead cells. 

I would suggest: Cell Counting together to 3H-thymidine or EdU assays. You could check the metabolism by CCK8 which is an indirect method to study the proliferation.

As an alternative to 3H-thymidine, CFSE staining of cells will give you an idea of cell proliferation - increased dilution of CFSE over time indicates the relative rounds of cellular division.

Trypan will only really give you a viability count.

Ki67 is another method you can use for cell proliferation by FACS analysis. This method is recommended to activated cells.

for cell proliferation you can use MTT or XTT methods.

Helloi Swapnil,

You need to be more specific on what your require from you flow facility, There are proliferation assays for flow assays utilising reagents like CFSE(488nm excitation), eFluor 670(635nm excitation) and eFluor 450(407nm excitation) with or without the addition of cell cycle staging agents like Brdu or the more surface co-labelling friendly Click-it system. If you have access to a cytometer with an absolute counting ability eg Miltenyi's Quant, BD Accuri C6, etc you can determine proliferation rates, cell cycle staging and phenotyping with the appropriate labelling procedures from a single tube. Best of luck.

Hello Martin,

Thank you very much for your kind inputs. Yah, I have access to BD Accuri C6 flow cytometer. Can you please brief me about its application for determination of  proliferation rates and cell cycle staging? 

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).

For those of you who could be interested by my own interpretation about “cytotoxic effects assessed by a colorimetric assay”, I strongly encourage then to read the attached document labeled QVM-10 Cytotoxicity ... As RG limits us to 10 attached files, I cannot attach here the 10 files that I am citing in the attached document QVM-10. If anyone among the RG community is interested by these attached documents, I can for sure send them.

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. And of course all the biochemical assays described by Galluzzi et al. (2009, 2012, 2015) referenced in the here attached QVM-10 document.

Best regards

Robert

Hi Swapnil, If you have an Accuri C6 this should be of use to you

https://www.bdbiosciences.com/documents/webinar-2014-02-Accuri-Apoptosis_K-T.pdf

if you have problems downloading I've a copy from 2014 on another computer.

best wishes

morning swapnil, This link to ThermoFisher has a selection of proliferation dyes that you should be able to multiplex

https://www.thermofisher.com/uk/en/home/life-science/cell-analysis/flow-cytometry/cell-health-and-viability-assays-for-flow-cytometry/cell-proliferation-assays-for-flow-cytometry/celltrace-reagents-for-cell-proliferation.html?

with cell cycle staging reagents like BrdU or ClikIt

https://www.thermofisher.com/search/results?query=clickit&resultPage=1&resultsPerPage=15&autocomplete=

Many thanks Martin

Dear Swapnil,

Who is "Martin"?

Best regards

Robert

Dear Robert,

Sorry, it is different.

Thanks to you for your inputs.

Regards,

Swapnil