difference between staining vital cell and dead - (Aug/29/2007 )
Hi, everyone, I want to stain my cell for fluorescence microscopy, I found that some protocol use 70% ethanol, and some not, I really wnoder why, is that depend on wether the cell you stain is live or not?
By the way , generally speaking, is there any difference between the staing for fluorescence microscopy and flow cytometry?
Thanks!
as far as i know the ethanol is used to fixate the cells. their membrans and outer proteins are denaturated. that makes them stick to the ground of the well. and of course it kills the cells.
and well generally speaking the fixation step is the difference between fluorescence microscopy and flow cytometry.
After the cells have been fixed, they all become dead cells.
70% EtOH is for fixation. It depends on a lot of things for people to use it or not.
1. If you are doing live image, then of course the fixation is out.
2. If you don't do live image, fluorescence microscopy would require fixation, then EtOH could be used as one fixing reagent. But the use of it will depends on your antigens, antibodies, or dyes. Some will not work with EtOH, some may only work with EtOH, and yet others are not so fussy. But, in general, speaking for myself, I found that PFA can be used with a broader range than EtOH. Even for cases that use alcohol for fixation, methanol, acetone are more common than EtOH.
3. I found people use EtOH more in flow cytometry. But even so, PFA could still be of use. It again depends on what you are staining and sometimes you have to try to find out.
4. For general IF, PFA is always my first choice.
Fixation can be used in either FM or FC, just depends on if you want cells live or not. You can stain cells, live or dead. But for live cells, in many cases, the intracellular structures cannot be stained, unless you can get the dyes inside the cells without killing them (some cell-permeabilizable dyes) or you have fluorescent proteins on those structures (you transfected the cells).
About flow cytometry (FC) and fluorescence microscopy (FM): I think you can see the difference from the names
- FC: no microscope, FM: obvious
- FC: may or may not include fluorescence, FM: again obvious
- FC: cells suspended in liquid, flow in single file across the light source, scattering lights which would then be collected by light detectors --> flow; FM: no flowing here.
- For application: FC would serve mostly in sort out the cells pools based on sizes, shapes, and in many cases also the fluorescent light from cells. That is, if you run a sample by FC, you will know how many cells are small, big, doublet...; how many cells that are stained. FM will give you the image of that fluorescent cells and show you where the signals are. For example, you transfect cells with GFP. Using FC, you know how many cells are green (got GFP). Using FM, you see for yourself that green cell and where it is green in the cells.
As you can see, since FC requires the cells to be suspended in liquid and flow in single file, cells cannot be fixed to a surface or clump to eachother. Live cells are quite common for FC, but if you fix them, you have to make sure that they are still suspended, not sticking to each other. While for FM, in most cases (except live image), cells are fixed immovably onto, say, coverslips. That is the main difference of staining cells between FC and FM
Hope that helps
1. If you are doing live image, then of course the fixation is out.
2. If you don't do live image, fluorescence microscopy would require fixation, then EtOH could be used as one fixing reagent. But the use of it will depends on your antigens, antibodies, or dyes. Some will not work with EtOH, some may only work with EtOH, and yet others are not so fussy. But, in general, speaking for myself, I found that PFA can be used with a broader range than EtOH. Even for cases that use alcohol for fixation, methanol, acetone are more common than EtOH.
3. I found people use EtOH more in flow cytometry. But even so, PFA could still be of use. It again depends on what you are staining and sometimes you have to try to find out.
4. For general IF, PFA is always my first choice.
Fixation can be used in either FM or FC, just depends on if you want cells live or not. You can stain cells, live or dead. But for live cells, in many cases, the intracellular structures cannot be stained, unless you can get the dyes inside the cells without killing them (some cell-permeabilizable dyes) or you have fluorescent proteins on those structures (you transfected the cells).
About flow cytometry (FC) and fluorescence microscopy (FM): I think you can see the difference from the names
- FC: no microscope, FM: obvious
- FC: may or may not include fluorescence, FM: again obvious
- FC: cells suspended in liquid, flow in single file across the light source, scattering lights which would then be collected by light detectors --> flow; FM: no flowing here.
- For application: FC would serve mostly in sort out the cells pools based on sizes, shapes, and in many cases also the fluorescent light from cells. That is, if you run a sample by FC, you will know how many cells are small, big, doublet...; how many cells that are stained. FM will give you the image of that fluorescent cells and show you where the signals are. For example, you transfect cells with GFP. Using FC, you know how many cells are green (got GFP). Using FM, you see for yourself that green cell and where it is green in the cells.
As you can see, since FC requires the cells to be suspended in liquid and flow in single file, cells cannot be fixed to a surface or clump to eachother. Live cells are quite common for FC, but if you fix them, you have to make sure that they are still suspended, not sticking to each other. While for FM, in most cases (except live image), cells are fixed immovably onto, say, coverslips. That is the main difference of staining cells between FC and FM
Hope that helps
Thank you for your detailed explaination!
So from where i can know that a dye is cytomembrane permeabilizable, is there a database like this?
Dye and permeabilizer are two different things. I am not sure if there are any that do both the functions.
The obvious thing is that dyes stain the cells while permeabiliser makes the cell membrane permeable so that the intracellular stains can go inside the cells. Saponin and Triton are some of the commonly used permeablisers. So, if U are doing some intracellular staining, you fix the cells with PFA,FA,GA, or EtH and then permeabilise the membrane and do the intracellular staining.
Another use of fixatives are if you want to store the stained cells. I have not done that but after staining if you want to preserve the cells and analyse but FCM later, then U can fix them with alcohol but the cells will be dead though staining will be preserved.
you can also fix with acetone, methanol or 50:50 acetone/methanol (i'd avoid GA as its pretty nasty - only really usefull in electron microscopy)
and you can perm with proteinase K (its in the middle of saponin/triton x)
dom
Maybe I did not make myself clear. What I meant is that there are dyes that are able to penetrate the membrane of living cells, thus they can stain the intracellular structures without the need to fix and permeabilize the cells --> stain and observe intracellular structures in living cells. I think that these dyes are considered cell permeabilizable (not permeabilizer, as in, they can get into cells (for them, cells are permeabilizable), but they do not make cells become permeabilizable to other things), but I may be wrong. If anyone know the proper name for them, please? Thanks in advance.
About the database for these dyes, I am not sure there is one. I would check out Molecular Probes catalogue, they have lots of dye, and they will inform you if those dyes are cell permeabilizable. Another way to check is to look for papers that have live image. They will tell you what they did. If they use any reagent that you need, you can find from them.
Almasy, I am sorry that I misunderstood the question. I was confused with the terminology used in the question when it was asked about 'cytomembrane permeabilizable'.
I now understand what U meant. U had given a very good description in your answer. Thank you so much for that. U meant dyes that are permeable to cytomembrane like CFSE while I was describing about permeabilisers. Confusion in terminology.
Are they called 'cytomembrane permeablilisable' or just 'membrane-permeable dyes'? 'Permeabiliser' will of course mean one that will make the membrane permeable.
I now understand what U meant. U had given a very good description in your answer. Thank you so much for that. U meant dyes that are permeable to cytomembrane like CFSE while I was describing about permeabilisers. Confusion in terminology.
Are they called 'cytomembrane permeablilisable' or just 'membrane-permeable dyes'? 'Permeabiliser' will of course mean one that will make the membrane permeable.
Yes, that's it. membrane-permeable
. You are right, THANKS a lot, Bungalow Boy. 
I must be getting old, muddled mind 