I used colony formation assay to test the IC50 in adherent cells such as MDA-MB-231 cell. However, I want to compare it with other breast cancer cells and some of them are suspension cells such as HCC2157 cell. But I do not know if there is colony formation protocol for suspension cells. Do anyone have suggestions? Or how to test IC50 of suspension cells?
Use MTT assay. centrifuge cell, count them, seed suitable concentration in each well of 96 well plate (suspended in 100 microliter) then add your drug in the other 100 microliter. incubate for 48 h then measure cell viability using MTT stain.
Use MTT assay. centrifuge cell, count them, seed suitable concentration in each well of 96 well plate (suspended in 100 microliter) then add your drug in the other 100 microliter. incubate for 48 h then measure cell viability using MTT stain.
Yes, Dr. Talib are rigth tratrazolium salts are simplest and very reliable... or you can use a dye exclusion staining like methylene blue (cheap, easy but tired) or propidium iodide by flow cytometer or fluorescence.
Yes Dr Talib and Dr. Yessica - MTT is the best way to overcome your problem .
Thank you, Dr. Talib, Yessica and Mainak. I will use MTT for my experiments.
You can use MTT or XTT assay. according to the results of mtt or xtt test you can calculate IC50 by using Calcusyn software.
We have successfully used methylcellulose cultures to perform in vitro clonal assays with cell lines that grow in suspension. The technique is not limited to leukemia/lymphoma cells. It also works well with many solid tumor cell lines, including the MDA-MB-231 line.
Thank you, Dr. Sieber! I think it is what I need! I will check the protocol.
See Miyagi et al, Journal of Photochemistry and Photobiology B: Biology 70 (2003) 133-144 for experimental details.
By the way, use Petri dishes, not tissue culture dishes, for clonal cultures anchorage independent cells. However, avoid Petri dishes that have been sterilized with ethylene oxide. View colonies with an inverted microscope, bright field (NOT phase contrast) with the condenser about two thirds closed. That will give you the depth of field required to easily count colonies.
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