How
to determine cell death?
Various assays exist to determine cell
death and all, unfortunately, have their limitations. In this section, I’ll
discuss all of the assays that I’ve got experience with. I’ll also indicate
whether I deem an assay suitable for high throughput screening (HTS) or not. In
general, I’d advise against using an assay that you don’t understand the principles
of. Companies such as Promega are never very eager to reveal the underlying
principles of their assays, as they’re afraid other companies will copy them,
but if you don’t know what, for example, the ‘live-cell protease’ activity is
that an assay such as Promega’s MultiToxFluor assay measures, you can’t possibly determine whether your treatment is indeed
affecting the cells’ health or just the activity of this mystery protease. If a
company won’t tell me what it is exactly that their assay does and what the
buffer components are, I won’t trust that assay.
MTT
assay
Figure 1. MTT assay to determine TNFa toxicity on L929 cells. |
A rather old fashioned method for
determining cell death is the good old MTT assay. This assay depends on the
reduction of tetrazole to formazan by oxireductase enzymes in living cells.
Formazan forms a purple precipitate that can easily be detected with an
absorption spectrometer. However, just like ATP assays, this assay also detects
loss of cells, lack of proliferation or reduced metabolic activity, rather than
cell death itself. MTT assays are certainly useful because they’re extremely
cheap and easy to perform, but shouldn't be used to accurately determine cell
death, let alone differentiate between modes of cell death. In addition, the
assay only really works well for adherent cells and the cells are lysed in the
process and the assay can therefore not be used in a multiplex set up.
Detection method: Absorption
Pros: Cheap and relatively easy
Cons: Sensitivity, not a cell death assay,
doesn't discriminate, samples destroyed in the process
Crystal
Violet
What I like about using crystal violet is
that the assay doesn't depend on metabolic activity of the cells, but
exclusively on the number of adherent cells remaining in your well. In
addition, the morphology of the stained cells can easily be judged by simple
light microscopy. Since the dye can be re-dissolved with methanol after
staining and washing, the assay can also be used to accurately quantify the
number of cells remaining in your well (Figure 2). Drawbacks are, of course, that the assay
can only be used for adherent cells, that the cells are fixed during staining
and that it’s hard to integrate the assay in a multiplex procedure. Since the
assay only determines loss of cells (which can also be lack of proliferation)
no conclusions about the mode of cell death can be drawn from crystal violet
staining alone. I find this assay particularly useful for illustrating colony
outgrowth after treatment, though it can also be used for routine screening.
Because the cell mono-layer easily gets damaged in the execution of the assay
during washing, fixing or staining, the assay is not very suitable for small
well formats. It works well enough in larger wells, down to a 24-well format,
since the relative contribution of some minor damage to the mono-layer won’t
influence the outcome of the assay as much in larger wells as in smaller wells.
Detection methods: Light microscopy,
absorption (after re-dissolving dye in methanol)
Pros: Very cheap and relatively easy,
objectivity
Cons: Sensitivity, not a cell death assay,
doesn't discriminate, only works for adherent cells, cells fixed in the
process, prone to errors when the cell mono-layer is scratched, toxicity of the
reagents
ATP
assays
Luciferase-based assay that determine
cellular ATP levels, such as Promega’s Cell Titer Glo, have become very popular,
especially in high throughput screens. Ease-of-use is the main selling point of
these assays, as they only require a single reagent addition and a short
incubation time. In addition, the assay is very sensitive. However, this assay
does not determine cell death. Rather, a reduction in ATP levels can reflect
several scenarios. First, a reduction in ATP levels in a given well might
indicate a reduction in cell numbers as a consequence of death, but could also
reflect a reduction in cell growth. If the treated cells expanded more slowly
than the control cells, this would be reflected in a relative reduction in ATP
levels. Second, overall ATP levels may be transiently reduced in cells under
stress without this resulting in cell death. Third, during apoptosis ATP levels
actually increase before cell death
occurs. Thus, cell death assays
based on determining relative ATP levels can very easily lead to invalid
results and wrong conclusions. For an initial screen, such an assay might be
useful but one should be extremely cautious to draw conclusions based on
results obtained from ATP assays alone.
Detection method: Luminescence
Pros: Easy, single addition, suitable for
HTS, very sensitive
Cons: Not a cell death assay, doesn't
discriminate, samples destroyed in the process
Dye
exclusion
In many older papers ‘apoptosis’ is equated
with cell permeability for DNA-binding dyes such as propidium iodide (PI) or
ethidium bromide (EthBr). However, apoptotic cells only become permeable to
such dyes at a very late stage, while necroptotic cells lose plasma membrane
integrity much earlier. Thus, dye uptake is either a sign of necroptosis,
primary necrosis or late-stage apoptosis (Figure 3). Bear this in mind when reading older
papers and don’t make this mistake yourself. Of course, this is not only true
for old-fashioned dyes such as PI, but also for newer dyes such as the Sytoxdyes from Life Technologies (formerly Molecular Probes/Invitrogen). Although
dye exclusion by itself has limited usability, it’s a method that can easily be
combined with other methods in a multiplex assay, as I’ll discuss below.
Detection method: Fluorescence
Pros: Cheap and very easy, single addition
and no washing required, suitable for both adherent and suspension cells, many
dyes available in different colours, suitable for HTS
Cons: Only indicates necrosis or
necroptosis, not early-stage apoptosis
LDH
Release
Figure 3. Sytox green staining vs. LDH release from U937 cells treated with TNFa in the presence of increasing amounts of zVADfmk for 24 hours. |
When cells lose membrane integrity, their
content is spilled in the environment. This includes the enzyme lactate
dehydrogenase (LDH). Activity of this enzyme can easily be detected with a
commercially available colorimetric assay. The great advantage of this assay is
that only the culture medium of the cells is required and that the cells
themselves can therefore be used for other assays, for example Western blot or
FACS. I found this assay to be very similar in sensitivity to dye exclusion (Figure 3). Of course, just
like dye exclusion, the assay only determines the rate of necrosis which can be
either a consequence of necroptosis, primary necrosis or secondary necrosis in
late-stage apoptosis. An advantage over dye uptake is that you only need an
absorption reader for detection and these are usually cheaper than fluorescence
readers, although the assay itself is somewhat more expensive.
Detection method: Absorption
Pros: Cheap and very easy, single addition
to culture supernatant, cells can still be used for continued culture or other
assays, suitable for both adherent and suspension cells
Cons: Only indicates necrosis or
necroptosis, not early-stage apoptosis
Resorufin/Resazurin
The principle of this assay, sold as AlamarBlue or Cell Titer Blue, is the conversion of blue, non-fluorescent, resazurin
to red, fluorescent, resorufin by living cells in an oxidation reaction. The assay has several advantages: the
cells are not destroyed in the process, the results can be measured with either
a fluorometer (red) or an absorbance spectrometer (red/blue) and can easily be
combined with other assays that utilize different fluorescence wave lengths or
luminescence. In addition, the results are aesthetically pleasing (Figure 3).
Detection method: Absorption or
fluorescence
Pros: Fairly cheap (I only once got a 10 mL
sample of Cell Titer Blue and used it for years) and easy, single addition
followed by incubation and measurement, suitable for multiplex assays, suitable
for HTS, cells remain alive and intact
Cons: Indicates oxidative metabolism, not a
cell death assay, doesn’t discriminate
Annexin
V Binding
Probably my favourite apoptosis assay is binding
of fluorescently labelled Annexin V binding followed by flow cytometry (FACS).
Annexin V binds to phosphatidylserine (PS) which is exposed on the outer
membrane of apoptotic cells. PS exposure is a passive process and happens when
a cell’s active mechanism for retaining PS on the inside of the plasma membrane
(the enzyme family collectively known as ‘flippases’) is compromised. Flippases are
ATP-dependent enzymes that are sensitive to Ca2+. Thus, a drop in
cellular ATP levels or a rise in intracellular Ca2+ levels will
inhibit flippases and lead to PS exposure.
Not too much is actually known about the regulation of these flippases during apoptosis, but PS exposure certainly is a very accurate hallmark of early apoptosis. However, PS exposure isreversible, some cells (such as macrophages) constitutively bind low levels of Annexin V and PS exposure can occur under certain rare conditions (such as Barth syndrome) in the absence of apoptosis. Nevertheless, I’ve generally found Annexin V staining to correlate nicely with apoptosis. Of course, when the cell membrane integrity is compromised, Annexin V will also enter the cell and stain both sides of the membrane. Thus, high Annexin V staining alone can be an indication of either apoptosis or necrosis. Whatever the case, when a cell displays high Annexin V positivity something’s wrong. Annexin V binding can (and should) easily be combined with dye exclusion for accurate differentiation of (early) apoptotic cells and necrotic cells. Bear in mind that Annexin V binding is Ca2+-dependent and your binding buffer should therefore always contain ~2.5 mM CaCl2. If you wash away the Ca2+, the Annexin V will also fall off. Finally, living cells will constantly expose low amounts of PS that are actively transported back in side and therefore constant exposure of living cells to Annexin V will very slowly lead to the uptake of the Annexin V and the staining of the cells. However, if you keep the cells on ice, you effectively fix the plasma membranes and the PS levels in the outer membrane won’t change anymore, even if you leave the cells unfixed.
Figure 5. Membrane asymmetry in healthy cells versus PS exposure in apoptotic cells. An image I drew years ago. |
Not too much is actually known about the regulation of these flippases during apoptosis, but PS exposure certainly is a very accurate hallmark of early apoptosis. However, PS exposure isreversible, some cells (such as macrophages) constitutively bind low levels of Annexin V and PS exposure can occur under certain rare conditions (such as Barth syndrome) in the absence of apoptosis. Nevertheless, I’ve generally found Annexin V staining to correlate nicely with apoptosis. Of course, when the cell membrane integrity is compromised, Annexin V will also enter the cell and stain both sides of the membrane. Thus, high Annexin V staining alone can be an indication of either apoptosis or necrosis. Whatever the case, when a cell displays high Annexin V positivity something’s wrong. Annexin V binding can (and should) easily be combined with dye exclusion for accurate differentiation of (early) apoptotic cells and necrotic cells. Bear in mind that Annexin V binding is Ca2+-dependent and your binding buffer should therefore always contain ~2.5 mM CaCl2. If you wash away the Ca2+, the Annexin V will also fall off. Finally, living cells will constantly expose low amounts of PS that are actively transported back in side and therefore constant exposure of living cells to Annexin V will very slowly lead to the uptake of the Annexin V and the staining of the cells. However, if you keep the cells on ice, you effectively fix the plasma membranes and the PS levels in the outer membrane won’t change anymore, even if you leave the cells unfixed.
Detection method: Fluorescence
Pros: Accurate assay for apoptosis,
sensitive, easily combined with other assays
Cons: Indicates both early apoptosis and
necrosis, not suitable for HTS
Caspase
activity
Caspase activity can be determined in a
variety of ways and is a fairly reliably indicator of apoptosis. Of course, as
I mentioned in the first post of this series, caspases are not exclusively
activated during apoptosis and it’s not trivial to tell the activity of one
caspase accurately apart from the activity of another caspase. However,
caspase-3 activity in particular is certainly a hallmark of apoptosis. Thus,
you’ll always find caspase-3 to be very active in apoptotic cells, although
limited caspase-3 activation can occur in non-apoptotic cells. To determine
caspase-3 activity any assay containing the tetra-peptide substrate ‘DEVD’ will
do. I prefer Ac-DEVD-AFC over AMC labelled substrates, since they seem to be
more sensitive and AFC will also turn yellow when released from the DEVD moiety,
which can even be detected by absorption. Those are available as fluorescent or
luminescent assays but you can also buy the substrate and make your own lysis
buffer. The substrate is also somewhat cell permeable and can therefore be
added to cells before inducing apoptosis and then be used to kinetically
determine the increase in apoptosis in a well. However, such an approach is
more likely to detect late-stage apoptosis, when the plasma membrane of the
cells becomes compromised. In addition, the DEVD is also consumed by the
proteasome, so healthy cells will hydrolyse it very slowly. Therefore, rather than stating that you’re
measuring caspase-3 activity when using DEVD as a substrate, state that you’re
measuring DEVDase activity as you can’t be absolutely certain that the activity
you measure is derived from caspase-3. Still, a fluorescently labelled tetra
peptide substrate can easily be combined with dye exclusion and viability
(resazurin/resorufin) to determine whether your cells have become apoptotic or
necrotic after treatment.
If you want to obtain more accurate information about the particular caspase involved, you could consider using an antibody that only detects the cleaved for of the caspase, tag it with a fluorescent label and perform flow cytometry. However, only the executioner caspases (3, 6 and 7) require cleavage for activation and the available antibodies detect other proteins with the same cleavage site as well. These are often cleaved as a consequence of caspase activity (caspases like to cleave their own linkers and will cleave every other protein with the same epitope as well) which is why Western blots with active caspase antibodies will often show a large amount of bands. In flow cytometry or microscopy assays you get no information about the size of the proteins labelled and therefore no accurate information about whether you’re really looking at the caspase or a product of caspase activity. A better method to determine which caspase has been activated is to label all active caspases with a biotinylated substrate, such as bVAD, bEVD or bVEID, perform a pull-down with streptavidin beads and detect your active caspases on Western blot.
Detection method: Fluorescence,
luminescence or absorbance
If you want to obtain more accurate information about the particular caspase involved, you could consider using an antibody that only detects the cleaved for of the caspase, tag it with a fluorescent label and perform flow cytometry. However, only the executioner caspases (3, 6 and 7) require cleavage for activation and the available antibodies detect other proteins with the same cleavage site as well. These are often cleaved as a consequence of caspase activity (caspases like to cleave their own linkers and will cleave every other protein with the same epitope as well) which is why Western blots with active caspase antibodies will often show a large amount of bands. In flow cytometry or microscopy assays you get no information about the size of the proteins labelled and therefore no accurate information about whether you’re really looking at the caspase or a product of caspase activity. A better method to determine which caspase has been activated is to label all active caspases with a biotinylated substrate, such as bVAD, bEVD or bVEID, perform a pull-down with streptavidin beads and detect your active caspases on Western blot.
Figure 6. Active caspase detection in cell extracts. Extracts were activated by addition of cytochomre c and caspase activity was detected by hydrolysis of the substrate Ac-DEVD-AFC (A) at the indicated time points or active caspases were labeled with bEVD-AOMK, pulled down with streptavidin beads and analyzed on Western blot (B). Active caspase-6 and -3 could be detected. Caspase-8 is cleaved (by caspase-6) but not activated under these conditions. See van Raam et al. for further details. |
Pros: Accurate assay for apoptosis,
sensitive, suitable for multiplex, suitable for HTS
Cons: Indicates mostly late apoptosis, risk
of false positives
The
ultimate multiplex assay?
In a good multiplex assay, you want to combine
at least one parameter to detect necroptotic cells and one to detect apoptotic
cells. I generally prefer to combine Annexin V binding with dye exclusion on a
flow cytometer, as flow cytometry provides a very versatile platform and also
provides you with valuable information about cell morphology, besides fluorescence. I've personally come to
prefer FITC-labelled Annexin V (I get it from Bender Med, now eBiosciences) with Sytox Red. FITC fluorescence
and Sytox Red are excited by different lasers and there’s therefore no need for
compensation, while PI and FITC are excited by the same laser and their
fluorescent peaks are close together. However, this assay is less suitable for
HTS, although most steps can easily be automated. I know the Vandenabeele lab
has developed an assay wherein they combine Sytox Green with Ac-DEVD-AMC to
detect caspase activity. This seems to work well for them, although I’d combine
it with a viability assay in the form of Cell Titer Blue. Assays that can be
performed kinetically are always superior to end-point assays, but in the end
the use of inhibitors can provide you with the most accurate information.