Apoptosis detection by AO staining (Amino Orange Stain)

Summary

The dye stains the cells simultaneously, providing a better method for identifying apoptotic and necrotic cells

Principle

3,6-(Dimethylamino)acridine hydrochloride, molecular formula C17H19N3-HCl-ZnCl2, molecular weight 438.12g/mo1, is a kind of fluorescent pigment, its detection excitation filter wavelength of 488nm, the blocking filter wavelength of 515nm. it and the cell in the amount of DNA and RNA binding there are differences in the amount of fluorescence can be issued by a different color, and the amount of binding with the DNA is less hair It can emit fluorescence of different colors, less fluorescence with DNA and more fluorescence with RNA, and more fluorescence with orange or orange-red. The dye has membrane permeability, can pass through the cell membrane, make the nuclear DNA and RNA staining. Therefore, under the fluorescence microscope, acridine orange can be observed through the normal cell membrane, so that the cell nucleus is green or yellow-green uniform fluorescence; and in the apoptotic cells, due to the chromatin condensation or breakage for the size of the fragments, the formation of apoptotic vesicles, acridine orange to make it stained with dense and densely stained yellow-green fluorescence, or yellow-green fragments of the particles: and the necrotic cells yellow fluorescence is weakened or even disappeared. Acridine Orange AO is often combined with ethidium bromide EB.

Appliance

Detection of apoptosis

Operation method

Detection of apoptosis by AO staining (yaconite orange staining method)

Materials and Instruments

[Reagent] pH 4.8-6.0 Tritionx-100 in PBS buffer.
AO dye: dissolve AO dye in PBS containing 1% TritionX-100 at a final concentration of 6 μg/ml with Rnase 100 μg/ml and store away from light.
【Instrument】Fluorescence microscope

Move

Prepare live cell suspension 106 /ml;Take 95 μl of cell suspension, add 5 μl of AO staining solution to mix and avoid light for 10 min;Add 5ml PBS, centrifuge at 1500r/min for 5min, discard the supernatant, and repeat the washing 2 times;Resuspend the cells, aspirate a drop on a slide and seal the slide with a coverslip;

Caveat

Under the fluorescence microscope, 515nm excitation light was used for microscopic examination: AO-stained cellular DNA showed yellow or yellow-green color. When the cell is apoptotic, the staining is dense and intensely stained with yellow-green fluorescence or yellow-green fragments are seen.

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