Asante Red Calcium

[Moninger, Thomas]

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Hello Group,

Do any of you folks have experience with Asante Red Calcium you would like to share? This emission ratio dye looks like a promising non-UV alternative to indo (and fura).

http://www.teflabs.com/ion-indicators/calcium-indicators/asante-calcium-red/

Cheers,

Tom

Thomas Moninger [hidden email]<mailto:[hidden email]>
Assistant Director
University of Iowa Central Microscopy Research Facilities
www.uiowa.edu/~cemrf<http://www.uiowa.edu/~cemrf>
P Think Green before printing this e-mail!


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[Max Peters]

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Try asking the authors of this paper, they seem to be only who published
experiments using this indicator.
http://www.sciencedirect.com/science/article/pii/S0006349511004188
Btw, it indeed looks promising.

On Wed, Aug 31, 2011 at 5:47 PM, Moninger, Thomas <[hidden email]
> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hello Group,
>
> Do any of you folks have experience with Asante Red Calcium you would like
> to share? This emission ratio dye looks like a promising non-UV alternative
> to indo (and fura).
>
>
> http://www.teflabs.com/ion-indicators/calcium-indicators/asante-calcium-red/
>
> Cheers,
>
> Tom
>
> Thomas Moninger [hidden email]<mailto:[hidden email]>
> Assistant Director
> University of Iowa Central Microscopy Research Facilities
> www.uiowa.edu/~cemrf<http://www.uiowa.edu/~cemrf>
> P Think Green before printing this e-mail!
>
>
> ________________________________
> Notice: This UI Health Care e-mail (including attachments) is covered by
> the Electronic Communications Privacy Act, 18 U.S.C. 2510-2521, is
> confidential and may be legally privileged.  If you are not the intended
> recipient, you are hereby notified that any retention, dissemination,
> distribution, or copying of this communication is strictly prohibited.
>  Please reply to the sender that you have received the message in error,
> then delete it.  Thank you.
> ________________________________
>

--
Maximilian Peters
Hebrew University of Jerusalem
Department of Physiology
Group of Prof. Minke
Hadassah Ein Kerem
Jerusalem 91120
Israel

Phone: [phone]
Fax: [phone]

[Thomas Volksdorf]

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Hello Tom and everyone else who might be interested in the ACaR-dye,

I am currently trying to establish this dye in our laboratory. I've ordered
the 10 x 50 µg kit of the AM-dye and try to load keratinocytes with it. I
already managed to get some pictures using our 2-photon-microscope, but I'm
not really satisfied yet because the signal for the unloaded dye (that you
need to calculate the ratiometrics) is very very week, but on the other hand
seems to have a high background noise. Using enough laserpower to actually
get a signal leads to significant bleaching and cell destruction...
I have to try a different PMT-setup yet, and a colleague of mine told me to
try imaging the cells with a CCD-Camera , so that I don't have to worry
whether one PMT might be more responsive or something like this. I'll
hopefully have the time do these tests this month, and I can provide status
updates if you like.

Kind regards,

Thomas

[Kilgore, Jason-2]

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Thomas,

I don't know this dye, so I can't be sure, but are you certain you don't need to cleave the AM group for it to become active?

Jason

** no corporate affiliation with TefLabs **

Jason A. Kilgore
Technical Application Scientist
Molecular Probes Labeling and Detection Technologies
Cells Systems Division
 
T 1 800 955 6288 then option 5  or  541 335 0353 . F 541 335 0238
29851 Willow Creek Rd . Eugene . OR . 97402-9132 . United States
www.invitrogen.com/technicalsupport

 



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Thomas Volksdorf
Sent: Thursday, September 01, 2011 5:18 AM
To: [hidden email]
Subject: Re: Asante Red Calcium

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Hello Tom and everyone else who might be interested in the ACaR-dye,

I am currently trying to establish this dye in our laboratory. I've ordered
the 10 x 50 µg kit of the AM-dye and try to load keratinocytes with it. I
already managed to get some pictures using our 2-photon-microscope, but I'm
not really satisfied yet because the signal for the unloaded dye (that you
need to calculate the ratiometrics) is very very week, but on the other hand
seems to have a high background noise. Using enough laserpower to actually
get a signal leads to significant bleaching and cell destruction...
I have to try a different PMT-setup yet, and a colleague of mine told me to
try imaging the cells with a CCD-Camera , so that I don't have to worry
whether one PMT might be more responsive or something like this. I'll
hopefully have the time do these tests this month, and I can provide status
updates if you like.

Kind regards,

Thomas

[Thomas Volksdorf]

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Hello Jason!

Like any other AM-dye (at least as far as I know...), the AM-ester of the
ACaR is supposed to be hydrolised automatically by intracellular esterases,
so after you have incubated the cells with the dye you let them stay for
another 45-60 minutes and then the dye should be ready. So it says in the
datasheet, and the few experiments that have been done by other researchers
followed this procedure.
I also followed another procedure published by invitrogen to test the dye
for responsiveness - basically you hydrolise the ester in KOH. After that I
was able to get a signal from my solution, and the signal @ 650 nm increased
when adding calcium, so the dye itself works.

So at least theoretically the AM-ester shouldn't be a problem, but thank you
very much for trying to figure out why my signals aren't as good as they are
supposed to be :)

[Jurkevic, Aleksandr]

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Thomas,

AM conjugate of ACaR has QY 0.03-0.04 and molar extinction coef. 23,000. It may explain to some extend low brightness of this indicator. For comparison, fluo-4 has QY 0.14 and molar extinction coef. of 77,000. Unfortunately, fluor-4 is non-ratiometric...

We were interested in a dextran conjugate of ACaR, but the company told us that it will be available only next year.

Aleksandr


Aleksandr Jurkevic, PhD
Associate Director
Molecular Cytology Core
University of Missouri-Columbia
120 Life Sciences Center
1201 E. Rollins St.
Columbia, MO 65211-7310

Phone:    [phone]
Fax:           [phone]
website  http://www.biotech.missouri.edu/mcc/



-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Thomas Volksdorf
Sent: Friday, September 02, 2011 2:10 AM
To: [hidden email]
Subject: Re: Asante Red Calcium

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Hello Jason!

Like any other AM-dye (at least as far as I know...), the AM-ester of the
ACaR is supposed to be hydrolised automatically by intracellular esterases,
so after you have incubated the cells with the dye you let them stay for
another 45-60 minutes and then the dye should be ready. So it says in the
datasheet, and the few experiments that have been done by other researchers
followed this procedure.
I also followed another procedure published by invitrogen to test the dye
for responsiveness - basically you hydrolise the ester in KOH. After that I
was able to get a signal from my solution, and the signal @ 650 nm increased
when adding calcium, so the dye itself works.

So at least theoretically the AM-ester shouldn't be a problem, but thank you
very much for trying to figure out why my signals aren't as good as they are
supposed to be :)