Hello Tran,

DAPI stains all DNA in  your cells.  It is not an mitosis marker.  However, Histone H3 phosphorylation is a widely used mitotic marker.  This is commercially available from Cell Signaling.  The following reference  states "The mitotic pattern of H3 phosphorylation is different in plants (Kaszas and Cande, 2000). First, phosphorylation begins late in prophase where the chromosomes are already quite condensed and it is observed on discrete locations on some chromosomes only. Second, at the metaphase plate all chromosomes show histone H3 phosphorylation primarily in the pericentromeric chromatin, in contrast to mammalian metaphase chromosomes, which are phosphorylated along their entire lengths. Finally, pericentromeric H3 phosphorylation is maintained at anaphase and disappears at telophase. The initial phosphorylation of H3 in prophase observed on compacted chromo- somes argues against an important role of this modi®cation in chromosome condensation in plants. "

Histone H3 phosphorylation and cell division   Oncogene (2001)2o, 3021+302

Fabienne Hans and Stefan Dimitrov

Dapi can be enough for a trained person. However, even for a trained person I recommend phospho histone at Ser10 (PH3). alpha-Tubulin staining can also be useful to evaluate mitosis progression. In your case I would use DAPI and anti-PH3 staining. Keep it simple.    

For accurate Mitotic Index analysis by FACS, single cell suspension is required. However, MI can also be determined manually under a simple microscope. For this, please  read the following web material:

http://fac.ksu.edu.sa/sites/default/files/mitotic_index_pdf.pdf

We have used DAPI in combination with anti-histone H3 antibody and Click-iT for flow cytometry. However your current tissue aggregates are too large and not permissible for standard flow. I can't see why this staining combination would not work in microscopy, if you can't generate cellular material smaller than

Best answer is that of Vijay Kumar. I always do exactly as is described on the web material he indicated. 

Mitotic Index by DAPI staining is very straightforward, and looks like your images show condensed chromosomes that are in mitotic cells. These can be counted and compared to interphase cells where the DAPI will show a more uniform oval shaped nucleus. Others have suggested adding phosphoH3 staining, which is also useful.

Check out this protocol. It may help you out.

Dear Tran,

A more difficult question as may first appear.  As mentioned above, direct observation is perhaps the best.  Mitotic figures are readily identifiable but like all static indices can be confounded by changes in the denominator, so if the total number of cells changes the index may be misleading, as 1/100 gives the same index as 2/100 despite there being double the number of dividing cells.  For more detail see

Quantification of epithelial cell proliferation, cell dynamics, and cell kinetics in vivo.

Goodlad RA.

Wiley Interdiscip Rev Dev Biol. 2017 May 5. doi: 10.1002/wdev.274. [Epub ahead of print] Review.

PMID:

 28474479

Dear all:

I am grateful to read all above-comments. I agree with Smith and Coelho for utilising other techniques, but I have no choice due to my lab's power for such techniques, that why I chose mitosis index to follow cell cycle progression. In fact a large number of papers used mitosis index as tool to determine synchrony level of cell population. In the case of mine so far,  the trouble is that I can not clarify or judge specific phases of cells and count total cell number in term of given images/observations. As suspecting, it may be that DAPI staining procedures applied is not good working enough or troubles in handling microscope as well. 

Below I would like to re-show other images observed several days ago, which  I changed some steps in a applied staining procedure. I think these images may be better than former ones for clarifying cell phases in given population, but I am not self-confidence on my judgement about cell phase in the images. I am wondering that if it is enough to obtain mitosis index !? Please help me point out which cell is in mitosis phase, which is in interphase as an example!!!

Moreover, it is acceptable if I calculate mitosis index for the population in the images as following:

- to count all cells in image as  number of total cell

- to count number of mitosis cells, and divide the number of mitosis cell by number of total cell to get mitosis index.

Thank you!

P/S: Image 1 white and black is another expression of image 1 using photo software

Dear Tran, 

If you want to do quantification of microscope images I recommend you reading the following post on the CellProfiler community forum: http://forum.cellprofiler.org/t/quantifying-microscopy-images-top-10-tips-for-image-acquisition/4808

If properly acquired, your images can be readily quantified with open-source automated image analysis software such as cell profiler. 

In the case you are using DAPI as the staining and you want to correlate the pixel intensity of your DAPI channel with increased chromosome condensation (mitotic cells), I also recommend you to use your DAPI solution to a final concentration of 2 ug/mL and stain for at least 30 minutes in order to have an homogenous staining. 

Nevertheless, I agree with Paula Coelho that if you really want to be sure about your mitotic index you should use an extra staining such as Phospho-Histone H3 (Ser10). 

Best wishes for your experiments,

André

Mitotic Index

            Mitotic index is analyzed in order to understand the toxicity of chemicals on cell proliferation in different treatment schedules. To analyze the rate of mitosis, the number of chromosomal mitotic cells a total of 2000 cells are scored for each treatment schedule and control. Mitotic index is calculated by using formula - 

% MI = No. of cells in mitosis X 100 / Total No. of cells scored