At our laboratory we are aiming to standardize the in vitro micronucleus test and have just begun to collect the relevant literature about it. It would be very helpful if anyone could point out the critical phases of the experiment and the share with us some of the troubleshooting related to the same. Sharing materials regarding it is also welcomed. Will the choice of test system effect my results? will working with the human lymphocytes be easier than other cell lines? Any other suggestions we ought to take into consideration?
Hi, i try to answer your question. MN test is very important and easy to obtain results. First you shoul add cytochalasin B to arrest cytokinesis at 44. h. And also you should add formaldehyde to the 3. Fixation step to preserve cytoplasm. Human lymhocytes is much more suitable than other cell type.Your results can be change according to the cell ttype since different cells posses different response.
Yes i understand the fact that different cell lines respond differently Mr Serkan Yilmaz but my query is that, why use CHO cell lines when we have human lymphocytes to work with? Isn't it easier than handling the complex CHO cell lines? Or is there some drawback that i am missing?
Yes handling of cells (frozen stocks) are easier than human lymphocytes. Also the other reasons are the lack of variation (it can occur in human lymphocytes) and large historical database. However CHO cell lines have some disadvantages at the genetic stability level.
Which cell lines do you use for micronucleus test? What are disadvantage and advantage?
I used HepG2 cell line for the in vitro MN assay as studies have shown the cells are sensitive to many genotoxins, also because HepG2 cells are from human origin and possess some metabolic capacities..
@Selma Urek: The cell lines we are planning to use are human lymphocytes.
@Noorasyikin Shamsuddin: Thank you for the input. Are the HepG2 cells stable when cultured frequently because we are into commercial tests. The guidelines of OECD suggest the use of the human lymphocytes.
@revathi dhanasheker. Could you please share with me the protocol of MN test in human lymphocytes?thank you in advance.
Yes, however certain studies that used HepG2 advised not to use more than 20 passage-old culture. In my experiment, I incubated HepG2 with cytochalasin B for 48 hrs since binuclear cells are not formed within 24 hours of incubation.