How to measure apoptosis / necrosis in a cell culture system?

Question

I'm trying to think of the simpliest way - perhaps some sort of assay from the supernatant? Any help would be much appreciated!

Answer 1

There are lots of apoptosis assays available.

One good assay is the TUNEL (Terminal dUTP Nicked-End Labelling) assay, which can be used with FACS or in fluorescent microscopy.

It uses a DNA ligase enzyme to conjugate tagged oligonucleotides to the nicked-ends of apoptotically-cleaved DNA in apoptosing cells. This therefore specifically labels the nuclei of apoptotic cells.

It is available from Boeringer as the "In Situ Cell Death Detection Kit" - which is what I use.

Another assay based on DNA cleavage is the "comet assay". You embed your cell suspension in agar, permeablise their membranes; you then perform electrophoresis on the gel, and stain with ethidium bromide. The cleaved DNA in apoptotic cells should electrophoretically move out of the cells in a "comet-tail", while the intact DNA in non-apoptotic cells is relatively immobile.

Apoptotic cells also display phosphatidylserine on the outer leaflet of their plasma membranes, due to the deactivation of "flipase" enzymes which normally keep it on the inner leaflet. This is an important in vivo "label" which allows phagocytes to recognise and consume them. Labelled annexin will bind to this phosphatidylserine, so can be used as a stain for FACS and fluorescent microscopy.

There are also several apoptotically-expressed proteins that can be stained for (like activated caspases, or caspase-cleaved proteins like PARP) by immunohistochemistry. Again - FACS or microscopy can detect this; or if you lyse the cells and extract the proteins, a Western-blot will detect them.

The simplest and cheapest (but most laborious) method would be to stain your cells with a DNA stain like DAPI or Hoescht. This labels ALL cells - but apoptotic cells have nuclei which are "pyknotic" (the chromatin has collapsed, and the nucleus has shrunk). These have to be identified by manual counting, unfortunately - but this is very do-able.

For necrosis, membrane permeation assays work well. Trypan blue or nigerosine should be excluded by live cells, but will penetrate dead cells.

A similar assay (the "LIVE/DEAD" assay) is based on two fluorescent dyes - calcein AM and ethidium homodimer. Calcein enters all cells, and is enzymically processed by cytoplasmic enzymes in live cells - staining them green. Ethidium is excluded by live cells, but will enter dead cells and stain their nucleir red.

FACS or microscopy can be used to get the data.

Answer 2

Apoptotic cells share a number of common features, such as phosphatidylserine (PS) exposure, cell shrinkage, chromatin cleavage, nuclear condensation, and formation of pyknotic bodies of condensed chromatin. Necrotic cells exhibit nuclear swelling, chromatin flocculation, loss of nuclear basophilia, breakdown of cytoplasmic structure and organelle function, and cytolysis by swelling. A number of assays are used for characterizing and distinguishing apoptosis and necrosis. Morphological assays include trypan blue exclusion, differential staining, and Hoechst staining. Methods to detect chromatin cleavage include TUNEL assays for whole cells and paraffin sections, DNA fragmentation assays using whole cells, assays of total genomic DNA, analysis of DNA fragmentation by agarose gel electrophoresis, phenol extraction of DNA for analysis of fragmentation, a quantitative assay for DNA fragmentation, and detection of DNA fragmentation by pulsed-field gel electrophoresis.

A simple method, if you do not need to differentiate the two processes, would be a Trypan Blue exclusion assay.  Cells that take up the dye are either apoptotic or necrotic.  To read the results you can either count the cells using a microscope or use a spectrophotometer set to at 590 nm to quantitate the difference in trypan blue uptake between different experiments.

I personally prefer the Annexin V-FITC assay that detects PS exposure on apoptotic cells and allow you to differentiate them from necrotic cells using a propidium iodide counterstain.  You can then easily quantitate the proportion of apoptotic cells in your experiment using FACS (fluorescent assisted cell sorting).  While the procedure if very precise in its results it does require expensive reagents and a flow cytometer.

Answer 3

you could do a growth curve...

inoculate a broth and take a UV-Vis reading about every hour (using uninoculated broth as your blank) until exponential growth starts then take them more frequently so you get a close reading for when it stops and then take readings during the exponential death

You could research the organism you're working with to try to figure out how long after inoculation the peak in growth should occur.