Question

Describe an experiment to examine if cells in culture are undergoing apoptosis. You should include a microscopy method as well as a molecular/biochemical method, and describe expected results for each, with reference to the cellular events known to occur during apoptosis.

Answer 1

Apoptosis of cells can be examined using several methods ranging from using microscopy, colorimetric/biochemical, spectrophotometric techniques, etc.

Two of the most commonly used techniques are MTS assay (which is a colorimetric assay) and Annexin V -PI assay (the results of which can be observed using fluorescence microscopy).

1. MTS assay: -

This is a cell viability assay that works on the principle of reduction of MTS tetrazolium compound and generating a formazan product (soluble in the cell culture media that is used). This reduction is by NAD(P)H- dependent enzymes in metabolically active (or) viable cells.

Procedure: -

(I) Collect the cells from the cell culture dish and add them to a 96 well plate, 48 hours before doing the assay and keep thee well plate in the incubator.

(ii) On the day of doing the assay, prepare the mix of MTS and PMS (both available as powders) in the ratio of 1:20 (PMS: MTS) in PBS. Each well should have 20l of this mix. Prepare this mix in an Eppendorf/a 15 ml falcon tube, wrap it with aluminum foil (as it is light sensitive), and immediately use for the assay. Take the correct weight of MTS and PMS according to the stock concentration (given in the container by the manufacturer) and the total volume needed for the assay.

(iii) Wash the wells containing the cells with 100l to 150l per well of PBS (to not have any hindrance in calorimetric reading due to the presence of other constituents in the media) after decanting the media. Empty the PBS by tapping the plate upside down.

(iv) Now add 100l of colorless media to 3 wells (for comparison). Then add 20l of the mix to each well including the wells with only the colorless media.

(v) Wrap the 96 well plate with aluminum foil and incubate in the incubator for 30 minutes. Then take the absorbance reading using a microplate reader at an absorbance of 490 - 500nm.

Expected Result: -

The live cells will turn brown at 490-500nm. The higher intensity of brown (that is more the absorbance value) obtained in the well will signify that the cells are more viable in that well.

2. Annexin V - PI assay: -

During the early apoptosis, Phosphatidyl Serine (PS) residues are translocated from the cytoplasmic face of the plasma membrane to the cell surface (they are present inside the plasma membrane when cells don't undergo apoptosis).

Annexin V has a strong dependent affinity for PS and therefore can be used as a probe for detecting apoptosis (when tagged with a fluorophore like FITC, APC, Cy5 or Cy3, etc.). Annexin V is used in conjunction with a vital dye like Propidium Iodide (PI) conjugated with another fluorophore (different from Annexin V fluorophore and having emission and excitation spectra not overlapping with each other) for identification of late apoptotic and necrotic cells. Viable cells with intact membranes exclude PI, whereas dead cells are permeable to PI.

Procedure: -

(I) Prepare 10X Annexin V buffer in 50ml of sterile PBS using the following constituents :

• 0.1M HEPES (It should be sterile. So open inside cell culture cabinet only)
• 1.4M NaCl
• 25mM CaCl2

Filter the buffer using a sterile 0.22m filter before adding to cells (as the buffer is supersaturated due to salts). Then before adding to cells, prepare 1X buffer by taking 1ml from the above-prepared buffer and making it up to 100ml using sterile PBS (take 9ml sterile PBS and add 1ml of the above prepared 10X Annexin V buffer). Prepare this according to the total volume of the buffer needed for all samples (100l per sample).

(ii) Add the required amount of Annexin V (tagged with a specific fluorophore) to the buffer (5l of Annexin V peer sample). So, for example, if there are 28 samples, the total buffer solution with Annexin V needed is 2800l (of which 140l is Annexin V and 2660l is Annexin V 1X buffer).

(iii) Take the required amount of cells in Eppendorf tubes, centrifuge the tubes (at the recommended conditions for the cells) and pellet down the cells.

(iii) Add the 1X Annexin V buffer to the cells. Incubate in a dark place for 30 minutes and then centrifuge & pellet down the cells. Now add the required concentration of PI (as specified by the manufacturer), after dissolving in PBS to the cells. Similarly, incubate for 30 minutes and then observe the samples under a fluorescence microscope (settings adjusted according to the manufacturer's guidelines for the specific company's microscope ).

Expected Result: -

For example, if the Annexin V is tagged with APC and PI is tagged with PE Texas Red fluorophores, cells showing both APC and PE Texas red fluorescence have undergone both apoptosis and necrosis. The cells showing only APC fluorescence are apoptotic cells and the ones showing only PE Texas Red fluorescence are necrotic cells.