Live/Dead discriminator for flow

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Amtekoth
Amtekoth's picture
Live/Dead discriminator for flow

 Hi,
I use a BD LSR II for multicolor flow.  I am using the 355nm laser for DAPI Live/Dead, but that has detector overlap issues if I'm also using the 405nm for Pacific Blue.  As I understand it,  Hoechst has the same problem.
I also use the 488nm for FITC, PE, PerCP-Cy5.5.
I also use the 633nm for Alexa Fluor 700,  and APC-Cy7.
So, does anyone out there make a good live/dead discriminator that falls in an uncovered detector range, perhaps around where PE-Texas Red or APC or PE-Cy7, or Pacific Orande or AmCyan fall?  Those are my only flexible detectors right now.
Thanks,
Ed

Jen_Floyd
Jen_Floyd's picture
 Propidium Iodide shouldn't

 Propidium Iodide shouldn't pose a problem.  From Wikipedia:
"Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used tostain DNA. When excited by 488nm of laser light, it can be detected with 562-588nm band pass filter. Flow cytometry is used to evaluate cell viability or DNA content in cell cycle analysis.[1] It can be used to differentiate necroticapoptotic and normal cells.[2]"

Amtekoth
Amtekoth's picture
Jen_Floyd wrote:

Jen_Floyd wrote:

 Propidium Iodide shouldn't pose a problem.  From Wikipedia:
"Propidium iodide (or PI) is an intercalating agent and a fluorescent molecule with a molecular mass of 668.4 Da that can be used tostain DNA. When excited by 488nm of laser light, it can be detected with 562-588nm band pass filter. Flow cytometry is used to evaluate cell viability or DNA content in cell cycle analysis.[1] It can be used to differentiate necroticapoptotic and normal cells.[2]"

 
Thanks for the info, Jen, but I thought PI interferes with PE.

Omai
Omai's picture
PI definitely bleeds over

PI definitely bleeds over into the FITC emmision spectra. It has a very broad emission peak. You can use Sytox red from molecular probes (invitrogen). Its only membrane permeable in dead cells. The "red" version excites with a 633 and emits at 660 (around where Cy5 emits). They also sell sytox blue and sytox green with different excitation and emission spectra. Maybe one of these will work for you.
(If you want to use PI, you could compensate for the bleed over into your FITC channel).
Omai

Jen_Floyd
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 Sorry about that.  I missed

 Sorry about that.  I missed the PE.  
Looks like Invitrogen might have something useful.  They have a collection of live/dead cell stains for FACS in just about every color.
http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/LIVE-DEAD-Viability-Brand-Page/LIVE-DEAD-Cell-Viability-Selection-Guide.html#intracellular

Amtekoth
Amtekoth's picture
Jen_Floyd wrote:

Jen_Floyd wrote:

 Sorry about that.  I missed the PE.  
Looks like Invitrogen might have something useful.  They have a collection of live/dead cell stains for FACS in just about every color.
http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/LIVE-DEAD-Viability-Brand-Page/LIVE-DEAD-Cell-Viability-Selection-Guide.html#intracellular

 
Thanks for the link.  I played around with their  SpectraViewer and found that the LIVE/DEAD® Fixable Red Dead Cell Stain Kit might work.  I'll contact their tech support and run it by them.

Thanks again,
 
Ed

Amtekoth
Amtekoth's picture
Amtekoth wrote:

Amtekoth wrote:

Jen_Floyd wrote:
 Sorry about that.  I missed the PE.  
Looks like Invitrogen might have something useful.  They have a collection of live/dead cell stains for FACS in just about every color.
http://www.invitrogen.com/site/us/en/home/brands/Molecular-Probes/Key-Molecular-Probes-Products/LIVE-DEAD-Viability-Brand-Page/LIVE-DEAD-Cell-Viability-Selection-Guide.html#intracellular

 
Thanks for the link.  I played around with their  SpectraViewer and found that the LIVE/DEAD® Fixable Red Dead Cell Stain Kit might work.  I'll contact their tech support and run it by them.

Thanks again,
 
Ed

Nope, the LIVE/DEAD Red spilled over into too many other channels that I needed.  I'm going to try their Live/Dead Blue.  I'm hoping it doesn't mess with my Pacific Blue too much...

heehawmcduff
heehawmcduff's picture
The reps from Molecular

The reps from Molecular Probes (Invitrogen) have been extremely helpful to me in the past and their R & D department is apparently working on new live cell/dead cell stains at the moment.  I would get in touch with them and see if they can offer any advice or perhaps a small sample of one of their new products.
Regards