===== Apoptosis and Cell Density Macro=====

====Installation and Use ====
Author: Ruben K. Dagda, Ph.D.
University of Nevada School of Medicine, Department of Pharmacology.
Introduction:
This is a macro for the quantification of both cell density and apoptosis by analyzing nuclear morphology in DAPI-stained fixed cells at 10X magnification.
It is well known that nuclear fragmentation, condensation and pyknosis are hallmarks of apoptosis. Although many cytotoxic stimuli can reduce cell numbers by causing cell detachment and/or necrosis, not all toxic insults promote apoptosis. To this end, the purpose of this macro is to quantify both cell numbers and apoptosis within the same image. This method may preclude the need to perform sequential MTT assays and propidium iodide staining as this macro provides cell number and percentage of nuclei exhibiting apoptosis.

This macro works with Image J versions 1.46 and higher.

You can go ahead and download the macro by clicking on the following link: {{:plugin:acquisition:dual_cell_density_apoptosis_macro:apoptosis_cell_count_macro_f2.txt|}}

Save the text file in the "Macro" folder of the ImageJ directory, install the imacro in Image J and press function 9 to start it.

=====Recommendations Before Starting=====

- Only use 10X magnifiction DAPI-stained images - Images have to be taken at very good intensity and contrast - Images can be analyzed by macro in color (Color Threshold function)

Limitations: - The color threshold functions can sometimes not separate some clumped nuclei efficiently.


=====Commands and Steps=====

The macro is simple. It will open up RGB images of DAPI-stained nuclei pressing function 9. Pressing Function 8 will then “Color Threshold” the images using an optimized function for DAPI stain. Pressing Function 7 will then count the number of nuclei and fragmented nuclei based on a certain area size. The macro will open up both the “Summary” window to give a total “Count” and a “Distribution Area” window which bins and counts the number of nuclei that exhibit a 3-fold reduction in average nuclear size. The “Count” field in the “Distribution Area” window is the number of apoptotic nuclei counted within the epifluorescence DAPI image.

The user can manually calculate the: Percentage of apoptotic nuclei as follows: Apotosis= ( the “count” given form the Distribution Area window)/ (by the total count from the summary window) X 100

The count within the Summary window is the total number of cells per epifluorecence field.

Make sure to close all the windows by pressing function 4 after finishing analyzing images. Make sure to close the “Color Threshold” window prior to analyzing another image.

=====ChangeLog and validation=====

Version: This is the first version of the macro. Any known fixes and updates will be published.

The macro was validated by the MTT assay in rat aortic smooth muscle A7R5 cells treated with the apoptosis inducer staurosporine (50% apoptosis) in the presence or absence of broad spectrum caspase inhibitors ZVAD (25% apoptosis).

In addition, it reduction in nuclear and increased circularity (nuclear area form factor) area is very well correlated with apoptosis induction as shown in the following study:

Iman M Helmy and Adel M Abdel Azim1 Efficacy of ImageJ in the assessment of apoptosis Diagn Pathol. 2012; 7: 15. Published online 2012 Feb 6. doi: 10.1186/1746-1596-7-15 PMCID: PMC3307432


=====Contact=====

Please direct any questions to Ruben K. Dagda, Ph.D. at: rdagda@medicine.nevada.edu


=====Funding=====

The maintenance and validation of the macro was supported by a Sanford Center for Aging Faculty Fellowship, a State of Nevada UNR Women Health Initiative grant and NIH grant NIGMS 103554.


=====License=====

The authors of the macro reserve the copyrights of the original macro. However, you are welcome to distribute, modify and use the program under the terms of the GNU General Public License as stated here: (http://www.gnu.org/licenses/gpl.txt) as long as you attribute proper acknowledgement to the authors as mentioned above.