Calcein AM should stain only living cells but it stains apoptotic bodies as well. What may be the reason? I use Jurkat cells
G'day Valentina --- This is not from personal experience note, however how about the following. POINT 1: The reason calcein AM is regarded as a "viability" stain is that it is membrane permeable in its esterified non-fluorescent form. Then inside the cell the various esterases lop off the ester generating fluorescent free acid species. Which is not membrane peremable, and so accumulates with the cell. POINT 2: So what calcein AM is really detecting is "esterase within a membranous container". And USUALLY this correlates with "viability" ... but not necessarily. POINT 3: So if the apoptotic bodies are still membrane bound, and still contain an esterase, then they will stain.
What do people think about that?
Bye --- Richard
G'day Valentina --- This is not from personal experience note, however how about the following. POINT 1: The reason calcein AM is regarded as a "viability" stain is that it is membrane permeable in its esterified non-fluorescent form. Then inside the cell the various esterases lop off the ester generating fluorescent free acid species. Which is not membrane peremable, and so accumulates with the cell. POINT 2: So what calcein AM is really detecting is "esterase within a membranous container". And USUALLY this correlates with "viability" ... but not necessarily. POINT 3: So if the apoptotic bodies are still membrane bound, and still contain an esterase, then they will stain.
What do people think about that?
Bye --- Richard
Thank you for these notes, Richard. I'm afraid calcein realy is not very good for the detection of apoptosis. Probably, esterases are not degraded or released during apoptosis and calcein doesn't mark only cells with injured membrane..
Check out the Current Protocols in Cytometry paper "Assessment of Cell Viability" - PMID 23546778. We compare a number of live/dead dyes and assays. Calcein AM is definitely not a good apoptosis stain. None of the live/dead stains are, really. You may want to try something like annexin V staining. It binds to externalized phophatidylserine. You can combine this with live/dead dyes to distinguish dead cells, live apoptotic cells, and live cells.
In addition to the interesting comment provided by Richard Horobin, I remind you that once Calcein AM is trapped inside the cell and activated by the esterase mechanism it will glow in the presence of free calcium. This means that although it is an apoptotic body, it could still have free calcium in the cytoplasm and maybe even some calcium management.
This also indicates that you may have applied Calcein early enough so the cells could have processed the AM part of the molecule before apoptosis, thus there is your fluorescence signal in the apoptotic bodies. If you delay Calcein incubation to the beginning of apoptosis you may circumvent that.
Good luck!
I have the same experience as Valentina - Calcein stains small vesicles detached from living cell. And I fully agree with Mr. Horobin.
Sometimes I can see more intensively stained regions inside the cell, they are small and bounded - I thought, this could be some membrane-bounded compartment (like endosomes etc.) which contains esterases...
I would be gratefull for your opinion.
Hi Ivana, yes "compartmentalization" is a well known artefact with many cytosolic calcium probes. We've recently writtten on this, so I'll attach a pdf ... Let me know what you think, mm? Richard Horobin.
Mr Horobin,
Thank you for the paper (and sorry for such late answer - I needed time to concentrate, read it and understand it...). It was very usefull to me, particulary the scheme Fig.6. Now I see, what will possibly happen to staining dye when it enters the cell and why the staining isn´t homogenous within the cell.
Recently I came across the paper from Tenopoulou et al. (2007) (it is cited in your work), where calcein depositing in lysosomes was discussed. Here was also mentioned autophagy, an interesting topic I thing. So this paper also helped me to understand the problem a lot.
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