We are looking for molecules whose presence is unique to arrest in G2 or M phases of cells cycle, whether as a result or as a cause of this arrest. We need to detect a molecule evidencing arrest as such, independent of its consequences or causes (i. e. DNA changes or cell death). It would be ideal to have proteins that can be dtected intracellularly by flow cytometry.
Propidium iodide staining and analysis by flow cytometric analysis gives information about arrest in G2 or M.
Sercan
anti MPM2 (mitotic cdk phospho epitope) staining by facs analysis, will detect M phase arrest!
G2 arrest ...bit more difficult! by westernblot..anti Y15 cdc2 probing!
don't know if these can be use for facs analysis
Thanks Sercan, Didier and Jan.
The Fucci system looks interesting, Jan. Do you know if it is amenable to flow cytometry?
I agree and not hesitate to Dr Sercan Ergün .
Multicolor flow analysis provides approaches to analyze proliferation
and differentiation potentials of cells and to localize cells
in different cell cycle stages.
About FLUOROCHROMES:
-Draq5 or Propidium iodide staining.
We use stainning with propidium iodide (PI) in B-ACUTE leukemias B-ALL: CD19-CD22-CD22-CD10/IP for exampleCycloscope™ kits , suitable for the evaluation of DNA content on neoplasic B-cell precursors in B lineage Acute Lymphoblastic Leukemia (B-ALL) samples in FACS cantoII..
Best regards
I'd agree to Dr. Didier Fesquet with anti-MPM2. PI staining is good for separating 2n and > 2n chromosomes, but that cannot tell the cell cycle phase G2 and M from S. Fucci will be the same. Both methods following chromosome copies cannot tell whether the cells are in G2/M arrest, unless you add another factor such as p53 or checkpoint proteins which is differentially expressed during arrest condition.
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