Please find the following protocol:

Quantitative Fluorescent Assay for the Simultaneous Identification of Apoptotic and Necrotic Cells


Overview:

The purpose of this fluorescent method is to simultaneously measure apoptotic cells, necrotic cells, normal (non-apoptotic/non-necrotic) cells, and apoptotic cells that have undergone secondary necrosis. Initially, apoptotic cells that show chromatin condensation will exclude Trypan Blue, Eosin Y, Ethidium Bromide or Propidium Iodide. Secondary necrosis is a cell culture phenomenon, since apoptotic cells in vivo are quickly phagocytized by macrophages or adjacent epithelial cells before they show extensive membrane damage.


Procedure:

1. Make a suspension of cells to an approximate concentration of 5 X 105 to 5 X 106 cells/ml.

2. Place 1 μl of Acridine Orange/Ethidium Bromide Dye Mix in bottom of a 12 x 75 mm glass tube.

3. Add 25 μl of the cell suspension and mix gently by hand.

4. Place 10 μl of this mixture on a clean microscope slide and cover it with a 22 mm2 coverslip. Although a hemacytometer may be used for visualization, the ordinary slide/coverslip combination results in a greater flattening of the cells than can be obtained with the rigid coverslip used with hemacytometers, making apoptosis more readily apparent.

5. Examine the sample using a 40X dry objective lens using epiillumination and a fluorescence filter combination suitable for observing Fluorescein.

6. Count a minimum of 200 total cells under the fluorescent microscope and record the number of each of the following four cellular states:

NOTE: It is extremely important to acquaint oneself with normal chromatin distribution in the cell line being studied. Both live and dead non-apoptotic cell nuclei will fluoresce green and have "structure", since variations in fluorescent intensity reflect the distribution of euchromatin and heterochromatin. Apoptotic nuclei, in contrast, have highly condensed chromatin that is uniformly stained by the Acridine Orange. This can take the form of crescents around the periphery of the nucleus, or the entire nucleus can be present as one or a group of featureless, bright spherical beads. In advanced apoptosis, the cell will have lost DNA or become fragmented into "apoptotic bodies" and the overall brightness will be less than that of a normal cell.

Viable cells with Normal Nuclei - bright green chromatin with organized structure

Necrotic Cell - bright orange chromatin with organized structure

Apoptotic Cell (intact membrane) - bright green chromatin which is highly condensed or fragmented

Apoptotic Cell (damaged membrane) - bright orange chromatin which is highly condensed or fragmented

7. Determine the percentage apoptotic cells and the percentage of necrotic cells:

Percent Apoptotic Cells = (number of apoptotic cells with and without loss of membrane integrity / Total cells counted) X 100

Percent necrotic cells = (number of non-apoptotic cells with loss of membrane integrity / Total cells counted) X 100


Solutions:

PBS
pH 7.2
2.7 mM KCl
4.3 mM Sodium Phosphate Dibasic (Na2HPO4)
1.8 mM Potassium Phosphate Monobasic (KH2PO4)
137 mM NaCl

Acridine Orange/Ethidium Bromide Dye Mix
Prepare in PBS
100 μg/ml Acridine Orange (CAUTION Biohazard!
100 μg/ml Ethidium Bromide (CAUTION Biohazard!)


Bioreagents and Chemicals:

Potassium Phosphate, Monobasic
Sodium Phosphate, Dibasic
Potassium Chloride
Ethidium Bromide
Sodium Chloride
Acridine Orange


References and Links:

1. Duke RC and Cohen JJ, "Morphological and Biochemical Assays of Apoptosis"; In: Current Protocols in Immunology, edited by Coligan JE, Krvisbeek AM, Margulies DH, Shevach EM and Strober W; New York: Green Publishing and Wiley-Interscience, pp. 3.17.1 - 3.17.16, 1992.

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