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Document: Apoptosis detection by FACS | Last modified: October 11, 2005
Detection Of Cell Viability And/Or Apoptosis By Flow Cytometry (FACS)
SP, September 2005

Viability, apoptosis, necrosis, death

Viable cells are cells that when allowed to continue beyond the timepoint of examination will stay alive. Besides live and healthy cells, cells in early stages of apoptosis may be considered viable as early apoptosis is believed to be reversible if the conditions inducing apoptosis are removed. Conditions inducing apoptosis include withdrawal of or exposure to certain factors (e.g., withdrawal of NGF, or exposure to TNF-α). These conditions are presented in vivo 'normally' (e.g., during maturation of lymphocytes) or 'abnormally' (e.g., during viral infections), or in vitro with our choice (e.g., addition of campotheticin chemical to cell culture) or with us unaware of or ineffective against them (e.g., lack of growth factors during handling).

Apoptosis ultimately leads to death and this can take from some minutes to many hours. The early stages of apoptosis are characterized by changes to mitochondrial membrane potential and cell membrane asymmetry (but not increased cell permeability). Later stages are characterized by DNA fragmentation and loss of cell membrane permeability. In general, apoptotic cells shrink (and can break up into smaller apoptotic bodies) and have characteristic nuclear changes that are visible under an electron microscope.

Necrosis, unlike apoptosis, is not a step-wise, controlled phenomenon. It is caused by physical disruption of the cell by physical (e.g., heat) or chemical (e.g., low pH) means, microbial toxins (e.g., by causing increased cell permeability), etc. Early necrotic cells have relatively-normal looking nuclei but their cell membranes (and organellar membranes) are fragmented. With time, organellar (including nuclear) disruption takes place and the cells generally swell, eventually bursting (and releasing inflammation inducers). Apoptotic cells in the last stages can necrose too.

Cytometric characteristics of non-viable cells

A sample of cells being examined by flow cytometry (e.g., for expression of a certain antigen) will have some non-viable cells (arising because of apoptosis and/or necrosis - see above for reasons). Unless one is specifically measuring apoptosis, these non-viable cells should be excluded from examination by gating. This is because, for example, they may non-specifically bind a fluorescent secondary antibody, the antigen being detected may have been degraded, etc.

Non-viable cells tend to have reduced FSC (can be high in case of swollen necrotic cells) but higher SSC values. Because of membrane permeability increases, DNA (and depending on the reagent, RNA too) of dead cells, necrosing cells and cells in late apoptosis will bind reagents such as propidium iodide (PI; MW 668) and 7-actinomycin D (7-AAD; MW 1270) to fluoresce more.

Because of loss of cell membrane asymmetry (for example, leading to the exposure of phosphatidylserine because of its increased presence on the outer leaflet), dead cells, necrosing cells and cells in early stages of apoptosis can be detected by binding of annexin V (a phosphatidylserine-recognizing protein).

As apoptosis involves induction of caspases, it can also be detected flow-cytometrically using fluorogenic caspase substrates or anti-caspase antibodies. Loss of mitochondrial membrane potential can be detected with reagents such as JC-1 that is lipophilic and upon exposure to negative mitochondrial potential (of healthy cells) reversibly aggregates.

A note on annexin V

Positive annexin V binding but lack of staining with PI (or 7-AAD) can thus be used to discriminate early apoptosis from late apoptosis and necrosis. However, it should be kept in mind that increase presence of phosphatidylserine on cell membrane (thus, increased annexin V binding) can occur because of other 'physiological' reasons (e.g., upon exposure to galectins).

Similarly, increased permeability to PI can be a result not of necrosis or apoptosis but of something else (e.g., exposure to galectins).

Staining cells with PI or 7-AAD for flow cytometry

PI fluorescence is detected in the FL2 or FL3 channels of the cytometer. High values indicate non-viability. 7-AAD is detected in the FL3 channel. High values indicate non-viability.

7-AAD is more permeable than PI; so, over time more cells will stain with 7-AAD than PI, and apoptosing cells will stain with 7-AAD earlier than with PI.

7-AAD solution at 1 mg/ml is prepared by dissolving 1 mg in 50 ul methanol and then diluting the solution with 950 ul PBS. Store at 4°C protected from light - can be used for atleast 6 months.

PI is used at 2-5 ug/ml and 7-AAD at 5-10 ug/ml final concentrations. A few seconds of incubation is enough in case of PI, whereas for 7-AAD, 10 minutes at RT or upto 30 minutes on ice is the typical duration of incubation.

Hoechst 33342

The HO342 or Hoechst 33342 dye, unlike PI, is non-polar and diffuses readily into cells (alive or non-viable) and binds AT-rich DNA regions. However, viable cells retain it more, and thus stain stronger. It is used at 1-5 ug/ml final concentration. Cells are incubated for 20-30 min at 37 deg. Fluorescence, detected in FL1, requires a UV laser, however.

Notes

1. Cells exposed to fixatives, detergents and hypotonic solutions will stain more with PI or 7-AAD.
2. Cells exposed longer to PI or 7-AAD will stain more.
3. A cell population being studied may not represent the true population, and thus may show less incidence of apoptosis than is true. E.g., when harvesting adherent cells, floating cells (usually apoptotic), may have been discarded. Similarly, apoptotic cells may be lost during other procedures such as low-speed centrifugation because of smaller size.
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