Using confocal microscopy imaging technique is it possible to calculate the percent apoptotic index? In some research papers it is mentioned but up to what extent it is reliable and is it necessary to validate using FACS analysis?
You have to count manually this way, which you may want to do on a regular fluorescent scope first then use confocal to collect your publication quality images. You will quantify this by calculating the percentage of Annexin-V positive and PI-negative cells divided by the total number of cells. I have also used Acridine Orange to calculate apoptosis with the PI staining, let me know if you need a protocol. Flow is calculating by sorting and is not necessary. Also, something to think about, are you trying to differentiate between apoptosis or secondary necrosis? If you want to make sure your quantification is unbiased you can do a blind quantification.
Hello C.B, apoptotic index from regular fluorescent microscope images can be obtained either manually as Dr. Skylar pointed out or by using the thresholding tool in ImageJ for confocal quality images. Thus, you will be able to eliminate biases due to manual counting. Using thresholding, you will be able to single out Annexin-V positive cells (e.g. green) which shows different color from PI-negative cells (e.g. red). After thresholding you can use the particle analysis tool also that will count those specified cells. Thank you and have a great day.
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