Hi Dan,

DAPI do not penetrate intact plant cells. You have to permeabilize them by fixation (Carnoy or Ethanol) or with detergent (Tween-20 0.1% or Triton X-100 0.01%), unless plasma membrane is depolarized.

In tobacco cell suspension you can observe such a depolarization at early mitosis phases such as pre-prophase or prohase.

DAPI staining is also a marker for early steps of Programmed Cell Death, which include membrane depolarization, so DAPI positive cells could be dead cells.

To avoid dead cells counting you should stain an aliquot of suspension with 1-2µg/mL Propidium Iodide for 5 min, rinse twice with culture medium, permeabilize with Triton X-100 and stain with DAPI.

Cells that are PI positive are dead cells, and then you won't count them.

Dear Catrice,

Thank for your suggestions. I feel better than from those of that  you gave me, but I still confuse to start handling this work. Here, I show you pictures observed from slide under flourescent microscope, C1 observed with bright field and C2 with flourescent light with the same area. Slide preparation include as followings: Mix 10 ul cell sample + 10 ul DAPI 1 mg/ml+ 10 ul Gluraldehyde 2%, incubate in dark 5 min, and pipette all to slide and squash by filter paper, and then observe under microscope. Could you bless some words about this observation ? C5 as another 

Moreover, I don't know how to count total cell number exactly and representative due to cell aggreates, and identify mitosis phase cell as well Please

The protocol you're using is OK unless DAPI concentration which is too high (333 µg/mL !). 1-10 µg/mL is enough. Don't waste money!

Your photos seem overexposed, it is thus difficult to see details such as chromatine condensation. I guess only one telophase on C5 image, the upper couple of cells. Try to adjust exposure to have only an overexposed pixels rate

Hi Catrice,

Thank for your comments. Still some terms that I don't understand, such as : "Your photos seem overexposed ?", and "Try to adjust exposure to have only an overexposed pixels rate

Hi Catrice,

I have tried modifications on DAPI staining procedure to improve observation quality as you recommended. Here, again I want to show you some images that I observed. In fact, my purpose is to aim to count "mitosis index" of the cell culture to assess effective of synchronization treatments applied. I need to produce a synchronous culture for further analysis. However, in this case I am quite confusing about how can I identify cell nuclei in mitosis phase from interphase (G1, S, G2) through such images; especially identify nuclei in anaphase and telophase. Because  the nuclei are various shapes and sizes and nuclei overlaping as shown in the images, it is difficult to clarify between two single cells and cells in anaphase and telophase. Moreover, in term of counting nuclei in prophase and metaphase, I intend to consider the nuclei which display strong and full intensity (as in image C1). To make more clear difference between nuclei in prophase and metaphase from others, I used  imageJ software to do some improvements, such as convert original colour images into greyscale (black-white) and threshold (see image C1' , C1'' are original from C1).

Anyway, I am confusing with two big questions:

1- whether DAPI staining is working on counting mitosis index in fact. For this,some ones say YES, others say NO.

2- How to clarify mitosis nuclei from others.

Please bless to me some your experiences on the images and to my questions

Thank a lot