Dyes and stains: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
 
(3 intermediate revisions by one other user not shown)
Line 1: Line 1:
This information was collected from various online sources. It is for informational purposes only.
==Useful links==
*http://www.probes.com/handbook/sections/0801.html
*http://www.probes.com/handbook/sections/1502.html
*[http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Molecular_Biology/Nucleic_Acid_Electrophoresis/Product_Lines/Dyes_and_Stains.html Dyes, Loading Buffers, and Nucleic Acid Gel Stains from Sigma-Aldrich]


===DAPI===
==Notes==
*358/461
*double stranded nucleic acid dye, associates with the minor groove of dsDNA, preferentially binds AT clusters
*stains both live and dead cells
*can be excited with a mercury-arc lamp or with the UV lines of the argon-ion laser
*better photostability than Hoesht dyes
*blue-fluorescent


===Fluorescein diacetate===
===Live cell stains===
*fluorescein (494/518) is formed by intracellular hydrolysis of FDA
*bacteria can hydrolyze this
*leaks from cells
*fluorescein has a relatively high rate of photobleaching
*pH-sensitive fluorescence ref (pKa ~6.4) that is significantly reduced below pH 7
*relatively broad fluorescence emission spectrum (not good for multicolor use)


===Carboxyfluorescein Diacetate===
There don't appear to be many dyes that selectively stain live cells.  When people want to detect live cells, I think that what they do is the following:  "The principle of this approach is to use simultaneously a permeant (SYBR Green [more recently SYTO green]; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable."  From Applied and Environmental Microbiology, October 2001, p. 4662-4670, Vol. 67, No. 10. So most stains that people bill as live cell stains are only membrane permeant stains and therefore stain both live and dead cells.
*fluorescein is formed by intracellular hydrolysis of CFDA
*carboxyfluorescein contains extra negative charges (structure) and is therefore better retained in cells
*been used in bacteria
*CFDA is moderately permeant to most cell membranes with uptake greater at pH 6.2 than at pH 7.4
*Oregon Green 488 (496/524) carboxylic acid diacetate is a more photostable version and less pH sensitive
*timing issues with how long you have to wait for the compound to be hydrolyzed ?


===Propidium iodide===
There a number of dyes from [http://probes.invitrogen.com/handbook/sections/1503.html Molecular Probes] that are supposedly pretty good at staining live cells a particular color. 
*535/617
For example, their BacLight Bacterial Membrane Potential kit uses a dye DiOC2 that is green in all cells, but turns red upon concentration at the cell membrane if there is an active proton gradient.  Also, there are dyes that measure redox potential of live cells through reduction of [http://probes.invitrogen.com/servlets/product?item=34951 C12-resazurin] --[[User:Skosuri|Sri Kosuri]] 14:29, 29 Apr 2005 (EDT)
*nucleic acid dye
*preferentially stains dead cells
*excited by 488 laser
*red-fluorescent


===SYTO===
'''Question'''
*from [http://probes.invitrogen.com/ Molecular Probes] now owned by [http://www.invitrogen.com/ Invitrogen]
*nucleic acid dye
*membrane permeant
*series of blue-, green-, orange and red-fluorescent dyes
*says that better results are obtained in buffers without phosphate so no PBS?
*would require some optimization


===Acridine Orange===
I am interesting in selectively staining live E coli cells for flow cytometry experiments. Essentially, the experiment I would like to carry out is to be able to simultaneously detect live E coli cells and measure YFP expression from these cells (where not all live cells will necessarily be producing YFP). So I need a stain that preferentially stains live cells and is compatible with YFP (ie has a different emission spectra so it can be measured simultaneously).
*500/526 for DNA, 460/650 for RNA
*nucleic acid binding dye
*cell-permeant
*emits green fluorescence when bound to dsDNA (525 nm) and red fluorescence when bound to ssDNA or RNA (650 nm).
*more common for eukaryotes?


===Hoechst 33342===
From what I have read one of your SYTO dyes would be best. Your viability kits use SYTO 9 but this isn't compatible with YFP. Is SYTO 9 the best for visualizing live cells? Are there any others you would recommend?
*350/461
*double stranded DNA, minor groove–binding DNA stains
*cell-permeant nuclear counterstain
*excited with the UV spectral lines of the argon-ion laser and by most conventional fluorescence excitation sources
*emits blue fluorescence when bound to dsDNA.


===SYBR Green I===
'''Answer from Invitrogen'''
*nucleic acid dye, better for DNA
*membrane permeant
*excited by 488 laser
*fluoresces green (maximum at 521 nm)
*tends to be used as a gel stain more often


===SYBR Green II===
The Orange or Red SYTO dyes may be an option for you. However none of them are live-cell only stains. What happens is these dyes are a total cell stain, and then you combine them with a dead-cell stain to determine viability. If you are not concerned with these, I would consider the red dye to co-localize with your YFP, this will minimize any signal confusion you would get with the Green or Orange versions. If your Flow Cytometer only has a 488 nm laser, than you will have trouble, because neither the Orange or Red ones have very good 488 nm excitation.
*nucleic acid dye, better for RNA
 
*membrane permeant
'''Answer from Biotium, Inc.'''
*excited by 488 laser
 
*fluoresces green (maximum at 521 nm)
Another approach would be to stain dead bacteria with EthDIII. With flow cytometry, you can calculate what percentage of the total cell population are dead and what percentage are YFP-positive.
*tends to be used as a gel stain more often but also cited in flow cytometry

Latest revision as of 15:46, 10 March 2015

Useful links

Notes

Live cell stains

There don't appear to be many dyes that selectively stain live cells. When people want to detect live cells, I think that what they do is the following: "The principle of this approach is to use simultaneously a permeant (SYBR Green [more recently SYTO green]; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable." From Applied and Environmental Microbiology, October 2001, p. 4662-4670, Vol. 67, No. 10. So most stains that people bill as live cell stains are only membrane permeant stains and therefore stain both live and dead cells.

There a number of dyes from Molecular Probes that are supposedly pretty good at staining live cells a particular color. For example, their BacLight Bacterial Membrane Potential kit uses a dye DiOC2 that is green in all cells, but turns red upon concentration at the cell membrane if there is an active proton gradient. Also, there are dyes that measure redox potential of live cells through reduction of C12-resazurin --Sri Kosuri 14:29, 29 Apr 2005 (EDT)

Question

I am interesting in selectively staining live E coli cells for flow cytometry experiments. Essentially, the experiment I would like to carry out is to be able to simultaneously detect live E coli cells and measure YFP expression from these cells (where not all live cells will necessarily be producing YFP). So I need a stain that preferentially stains live cells and is compatible with YFP (ie has a different emission spectra so it can be measured simultaneously).

From what I have read one of your SYTO dyes would be best. Your viability kits use SYTO 9 but this isn't compatible with YFP. Is SYTO 9 the best for visualizing live cells? Are there any others you would recommend?

Answer from Invitrogen

The Orange or Red SYTO dyes may be an option for you. However none of them are live-cell only stains. What happens is these dyes are a total cell stain, and then you combine them with a dead-cell stain to determine viability. If you are not concerned with these, I would consider the red dye to co-localize with your YFP, this will minimize any signal confusion you would get with the Green or Orange versions. If your Flow Cytometer only has a 488 nm laser, than you will have trouble, because neither the Orange or Red ones have very good 488 nm excitation.

Answer from Biotium, Inc.

Another approach would be to stain dead bacteria with EthDIII. With flow cytometry, you can calculate what percentage of the total cell population are dead and what percentage are YFP-positive.

Retrieved from "https://openwetware.org/mediawiki/index.php?title=Dyes_and_stains&oldid=872108"