Since it is a very hazardous chemical I do not want to use it and also it is expensive too.
Hello Himanshi
CytB is used to restrict the cells observed to those that have divided once following treatment. In our lab we always use CytB when performing MN studies.
However, there are some papers communicating good results without it. In a paper published in Mutagenesis in 1999, Matsushima T et al consider that the CB cytokinesis block is not necessary for continuously growing established cell lines (http://mutage.oxfordjournals.org/content/14/6/569.long).
On the other hand, M. Fenech (in my oppinion the "father" of MN assay) says: "It is evident that the best way to eliminate any possible confounding effects of altered cell division kinetics is to use the CBMN Cyt assay regardless of the cell type used .....The choice not to use Cyt-B is justifiable only in those rare situations in which cytokinesis defects are being specifically investigated". (Nature protocols, 2007: http://www.nature.com/nprot/journal/v2/n5/full/nprot.2007.77.html ). More recently, in a paper published in 2011 in Mutagenesis, Fenech showsw that "A key advantage and highlight of the CBMNcyt assay is that it allows simultaneous detection of multiple molecular events that lead to chromosome damage and chromosomal instability", i.e. micronuclei, nucleoplasmic bridges and nuclear buds (http://mutage.oxfordjournals.org/content/26/1/125.long).
My personal oppinion is that CytB is mandatory in lymphocytes and very useful in oher types of cells. I also believe that its use depends on the study endpoints too. If your work aims to bring insights into the molecular mechanisms of cytotoxicity, then you should use CytB. But, if you plan to test only the behaviour of fibroblasts or the genotoxicity of some compunds and CytB is a problem, you could perform a MN assay without CytB and see what happens.
see the link for a basic info
http://www.crios.be/genotoxicitytests/micronucleus_test.htm
Dear Himanshi,
if you plan to study lymphocytes you must use cytochalasin, because this method takes into account micronucleus in binucleated cells (such cells you can get after the addition of cytochalasin).
No I am not performing it on lymphocytes but fibroblasts. What will you suggest now.
Hello Himanshi
CytB is used to restrict the cells observed to those that have divided once following treatment. In our lab we always use CytB when performing MN studies.
However, there are some papers communicating good results without it. In a paper published in Mutagenesis in 1999, Matsushima T et al consider that the CB cytokinesis block is not necessary for continuously growing established cell lines (http://mutage.oxfordjournals.org/content/14/6/569.long).
On the other hand, M. Fenech (in my oppinion the "father" of MN assay) says: "It is evident that the best way to eliminate any possible confounding effects of altered cell division kinetics is to use the CBMN Cyt assay regardless of the cell type used .....The choice not to use Cyt-B is justifiable only in those rare situations in which cytokinesis defects are being specifically investigated". (Nature protocols, 2007: http://www.nature.com/nprot/journal/v2/n5/full/nprot.2007.77.html ). More recently, in a paper published in 2011 in Mutagenesis, Fenech showsw that "A key advantage and highlight of the CBMNcyt assay is that it allows simultaneous detection of multiple molecular events that lead to chromosome damage and chromosomal instability", i.e. micronuclei, nucleoplasmic bridges and nuclear buds (http://mutage.oxfordjournals.org/content/26/1/125.long).
My personal oppinion is that CytB is mandatory in lymphocytes and very useful in oher types of cells. I also believe that its use depends on the study endpoints too. If your work aims to bring insights into the molecular mechanisms of cytotoxicity, then you should use CytB. But, if you plan to test only the behaviour of fibroblasts or the genotoxicity of some compunds and CytB is a problem, you could perform a MN assay without CytB and see what happens.
yes I just want to see the effect of my plant based drug on cancer cell lines using micronucleus assay
Hello Himanshi,
Very simply, yes you can absolutely perform the mn test without cytochalasin.
You will want to make sure you have a way to measure the cells numbers so that you will know if your cells are dividing. The simplest way is to look at relative increase in cell counts (RICC). See the publication below for a very good comparison of with and without CYB and the different cytotoxicity measurements.
Schuler, M et.al. Mutation Research 702 (2010) 219–229 Title is Evaluation of phenolphthalein, diazepam and quinacrine dihydrochloride in the in vitro mammalian cell micronucleus test in Chinese hamster ovary (CHO) and TK6 cells.
Good luck!
Liz Rubitski
Article Evaluation of phenolphthalein, diazepam and quinacrine dihyd...
I guess, CytB is most essential for lymphocyte analysis where it arrests cell cycle and creates the bi-nucleated cells and it is the bi-nucleated cells you count and an associated MN. But, I went across the paper shared by Rubitski. Its nice to know that there are some other parameters to assess. Thanks.
Well it depends to some extent on the type of cell line you use. To brief if used lymphocytes isolated from blood it for sure requires Cytochalasin B for differential blocking of cell cycle at cytokinesis stage.
On the other hand, if cell lines are used (viz., CHO, NRK-49F and so on...) it is not essentially required to use the cytochalsin. The procedure what we follow in our lab....
what are you suggested instead of CYTB in micronucleus assay?
I want to use lymphcytes isolated from blood for the genotoxicity .
Dear Madam,
Tou can use colshicin instead of cyto B. Just search papers used colshicin in micronucleus test and get the concentration of it.
When using human samples, M. Fenech created a protocol based on buccal mucosal cells, without CytoB tretament
https://www.nature.com/articles/nprot.2009.53
I use flow cytometry for MN scoring at my lab , there's no need at all of CytoB
Article In vitro micronucleus assay scored by flow cytometry provide...
Daniel Perez Vieira Hi Daniel, I was wondering how do you score micronuclei by flow cytometry?
All pertinent explanations are on this paper (Bryce SM et al, 2007); we use to follow the protocol with no modifications
Article In vitro micronucleus assay scored by flow cytometry provide...
Using DNA staining (SYOX) and membrane lysis, the protocol can discriminate nuclei (high FSC, high FL1) from micronuclei (low FSC, low FL1) through a simple gating method.
The protocol also uses a method to label non-viable cells (EMA - ethidium monoazide) prior to cell lysis to prevent from counting apoptotic nuclei fragments as MN, counting nuclei from dead cells.
Latex beads are added in same amount in all samples to give us a relative nuclei count ("nuclei-to-bead ratio"), in a way to surrogate the CBPI analysis.
other
Hope it helps!
Hi, Dr
you can check this thesis
Thesis UTILIZATION OF MICRONUCLEUS TEST AND GENETIC MARKERS FOR DET...
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