Self prepared material:
Fluorescence microscope; PBS; Cell counting board; Glass slide; Cover glass slides

1. Preparation of staining working solution
Before the formal experiment, take acridine orange staining solution (reagent A), ethidium bromide staining solution (reagent B), and dilution buffer solution (reagent C), and dilute them to the required AO/EB staining working solution in the ratio of A: B: C=1:1:8
2. Cell preparation
Adherent cells: Digest according to conventional methods. Centrifuge at 1000 rpm for 5 minutes, collect cell precipitates, and wash once with PBS6pieces/ml
3. Staining
Every 25μ Add 2&mu to the cell suspension; Freshly prepared AO/EB staining working solution, gently mix evenly. Incubate at room temperature for 5-10 minutes
4. Observation
Take a clean slide and add 5-10&mu dropwise; Cover the stained cell suspension with a cover glass. Observe and count using a fluorescent microscope's luciferin filter and a 60x magnification objective. This measurement should be repeated at least three times, with at least 100 total cells observed each time

  Acridine orange (AO) is a heterochromatic nucleic acid binding dye with cell membrane permeability. It binds to DNA or RNA by insertion or electrostatic attraction. When combined with double stranded DNA (dsDNA), it emits green fluorescence (ex/em=502/525nm), and when combined with single stranded DNA (ssDNA) or RNA, it emits red fluorescence (ex/em=460/650nm), with a small amount of binding showing orange red fluorescence. Ethidium bromide (EB) is a polycyclic aromatic hydrocarbon fluorescent small molecule and nucleic acid insertion agent, which is chimeric into the base pairs of double stranded DNA or RNA. It has no base specificity and shows red fluorescence ((ex/em=518/605nm). EB is not cell membrane permeable
  Acridine orange (AO) and ethidium bromide (EB) are often used in combination to observe the changes of nucleus and the formation of apoptotic bodies, which are the characteristics of apoptosis. The combination of the two can distinguish living cells, apoptotic cells and necrotic cells. The working principle is that acridine orange can stain both live and dead cells, while EB can only stain cells that have lost membrane integrity. The living cells are uniformly green. Early apoptotic cells are green, and bright green dots are seen in the nuclear energy of cells due to chromosome condensation and nuclear breakage. Late apoptotic cells will also be stained with EB and thus become orange red, but compared with necrotic cells, late apoptotic cells will have concentrated and often broken nuclei. Necrotic cells are also stained orange red, but will have a nuclear morphology similar to that of living cells, without condensed chromosomes.

Product Components:

1. It is also commonly used to double stain cells with H O echst 33342, which can produce blue fluorescence after binding to dn a through the living cell membrane, and propidiu m iodide, which can only produce red fluorescence after binding to dn a through dead cells
2. If there is a low-temperature centrifuge for centrifugation, the effect is better
3. During operation, pay attention to reducing the exposure time of reagent (a) and reagent (b) to strong light
4. EB solution has certain toxicity, please be careful
5. For your safety and health, please wear lab clothes and disposable gloves