Please find the following protocol:

Staining Apoptotic Cells with Propidium Iodide


Overview:

This protocol describes how to stain cells with Propidium Iodide for the detection of Apoptosis after they are treated with a desired agent. Propidium Iodide will intercalate into the DNA of the cells and will be visible under Fluorescent Microscopy. Apoptotic cells should be shrunken and have fragmented (pyknotic) nuclei with condensed chromatin.


Procedure:


Cells in Suspension

1. Seed cells in a 6-well dish at 2.5 X 105 cells per well with 2 ml of cell culture media per well.

2. Allow the cells to attach and grow for 24 hr and then treat with desired agent.

3. At the end of the desired time interval, collect the media from a well and divide the aliquot into two 1.5 ml microcentrifuge tubes. If preferred, detached cells can be stained separately.

4. Rinse the empty (cell-containing) well with 250 μl of PBS and add this PBS to one of the microcentrifuge tubes from Step #3.

5. Add 450 μl of Trypsin Solution to the well and incubate the plate at 37°C until the cells are fully Trypsinized.

6. Add 300 μl of serum-containing media to the well (Make sure the cells have completely detached). Recover the cell suspension from the well and add it to tubes from Step #3 (Balance the volumes in the 2 tubes from Step #3).

7. Centrifuge the tubes at 5,000 rpm in a microcentrifuge (i.e. at a low setting) and remove the supernatant.

8. Add 250 μl of Mg-Ethanol Solution to one of the tubes.

9. Gently vortex the tube to break up the cell pellet and then combine this cell suspension with the pellet in the duplicate tube and gently resuspend that cell pellet..

10. Add 0.1 volume of 1 mg/ml RNase to the cell suspension.

11. Incubate the sample for 1 hr at 37°C.

12. Centrifuge the cells at 5,000 rpm in a microcentrifuge (i.e. at a low setting) and remove the supernatant.

13. Add 100 μl of 500 ng/ml Propidium Iodide and gently break up the cell pellet. At this point, the samples can be covered in Aluminum Foil and stored at 4°C.

14. To visualize the cells, make sure the cells are in suspension (gently break up the cell pellets in necessary) and place 20 μl of the suspension on a microscope slide and add a coverslip.

15. View the cells under a fluorescent microscope. The peak excitation λ and emission λ for Propidium Iodide is 536 nm and 620 nm, respectively. The excitation and emission spectra are rather broad, so care must be taken in the selection of the second fluorochrome if dual labeling is desired.


Cells Grown on Coverslips

1. Seed the cells as in Step #1 - Cells in Suspension, but place a coverslip into the wells of the plate just before seeding so the cells can adhere to it.

2. Allow the cells to attach and grow for 24 hr and then treat with desired agent.

3. At the end of the desired time interval, remove the media from the wells of the plate. Save the media, if desired.

4. Add 2 ml of Mg-Ethanol Solution and 0.1 volume of 1 mg/ml RNase A.

5. Incubate the plate for 1 hr at 37°C.

6. Remove the liquid from the well.

7. Add 1 to 2 ml of 500 ng/ml Propidium Iodide for about 1 hr at 37°C.

8. Remove the liquid and place the coverslip onto a glass slide with mounting media.

9. View the cells under fluorescence microscopy. The peak excitation λ and emission λ for Propidium Iodide is 536 nm and 620 nm, respectively. The excitation and emission spectra are rather broad, so care must be taken in the selection of the second fluorochrome if dual labeling is desired.



Solutions:

Mg-Ethanol Solution
15 mM MgCl2
25% (v/v) Ethanol

0.25% (w/v) Trypsin Solution
Make up in PBS

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

500 ng/ml Propidium Iodide (CAUTION Biohazard!)

1 mg/ml RNase A



Bioreagents and Chemicals:

Sodium Phosphate, Dibasic
Potassium Chloride
Sodium Chloride
Ethanol
Trypsin
Potassium Phosphate, Monobasic
Propidium Iodide
Magnesium Chloride
RNase A


Reference Link:

http://www.bio.com/protocolstools/protocol.jhtml?id=p1987