I have studied some techniques of genotoxicity (micronucleus assay and mouse lymphoma-TK assay) and i should set up it in the laboratory. But there is not enough experiential instructions in my hands...may I ask to anyone who is familiar with these techniques to guide me?
thanks
Whether you want to try Blood micronucleus assay or liver micronucleus assay. If you have lack of set up then I would recommend Blood micronucleus assay. It just needs a simple staining solution and a laboratory microscope. There are multiple articles by Jena GB on pubmed, that you can refer for protocol.
Hi Dear Murli..
No No...
i cant use invivo tests...it should be invitro assay, using cell lines..
hi
i'm giving you some information on Micronucleus assay (MN) which is now emerged as Cytokinesis block micronucleus cytom assay (CBMN cyt Assay).
The best articles to read are Dr. Micheal Fenech's CBMN protocols and reviews on the inferences of test results!
pls refer below articles
1) Nature Protocols 2, - 1084 - 1104 (2007) doi:10.1038/nprot.2007.77
Cytokinesis-block micronucleus cytome assay
Michael Fenech1
2)Mutation Research 534 (2003) 65–75
HUMN project: detailed description of the scoring criteria for
the cytokinesis-block micronucleus assay using isolated
human lymphocyte cultures
M. Fenech W.P. Changb, M. Kirsch-Volders , N. Holland , S. Bonassi , E. Zeiger
3) Mutagenesis vol. 26 no. 1 pp. 125–132, 2011
Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud
formation in mammalian and human cells-M. Fenech, M. Kirsch-Volders, A. T. Natarajan, J. Surralles, J. W. Crott, J. Parry,y, H. Norppa, D. A. Eastmond, J. D. Tucker and P. Thomas
The above articles give us a basic knowdelge to perform CBMN cyt assay with a better understanding of the mechanism involved in the formation of end points (MNi, NPB and NBUDs), which may help to deliver some solid inferences on your study!
some more articles are there which m quoting below- may be useful
4) Environmental Health Perspectives Supplements Vol. 101 (Suppl. 3): 101-107 (1993)- The Cytokinesis-Block Micronucleus Technique and Its Application to Genotoxicity Studies in Human Populations.
Michael Fenech
5)Mutation Research 428 1999. 271–283
The HUman MicroNucleus Project—An international collaborative study on the use of the micronucleus technique for measuring DNA damage in humans
Michael Fenech, Nina Holland , Wushou P. Chang , Errol Zeiger , Stefano Bonassi.
6)Mutation Research 455 (2000) 81–95
The in vitro micronucleus technique
Michael Fenech
7)Mutation Research 404 (1998) 155 - 165
Important variables that influence base-line micronucleus frequency in cytokinesis-blocked lymphocytes a biomarker for DNA damage in human populations
Michael Fenech
the nature protocol explains the method for lymphocytes and cell lines, scoring criteria you can use for cell lines also
hope this could help you
Good Luck
Regarding your question I sent the following reference may be useful.:
Michael Fenech. Mutation Research 404 (1998) 155 - 165
I wish I would be helpful. Here is the protocolfor cell line...
Micronuclei study with cell cultures: Cells were treated for 24 h. After treatment,cells were further cultured with cytochalasin-B for 24 h.Then, cells were trypsinized centrifuged (800 RPM ) and resuspended in 0.075 M KCl (4 ml), and incubated for 2 min. Cells were then fixed 3 times in methanol:glacial acetic acid (3:1)(4 ml). After first fixation 6-7 drops formaldehyde were added. Following fixation, the cell solution was dropped onto pre-cleaned slides (let slides dry at least 1 hour) and the nucleus was stained by 5% giemsa for 10-15 min.
Dear Professor Nilufer.
Your kind reply helped me a lot...
I have found some positive controls in papers for this assay..such as Cytosine Arabinoside, Mitomycin, Colchicine and...but may i ask you what is the most common and safe material to use as a positive control for this assay ?
Is there any commercial material to use as a positive control for this assay?
We use hydrogen peroxide for positive control in MN tests..
Please see the attached file as an additional information
Article Antigenotoxic Potential of Rutin and Quercetin in Swiss Mice...
Dear Dr. you can check this thesis Thesis UTILIZATION OF MICRONUCLEUS TEST AND GENETIC MARKERS FOR DET...
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