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additional probes to standard lineup, Brilliant Violet?

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mcammer

additional probes to standard lineup, Brilliant Violet?

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One of our clients wants to add a fifth or sixth probe to the standard Dapi, AF488, AF568, AF647 lineup.

One of our confocals has a 594 nm laser and we also have true spectral detection, but rather than sandwich in overlapping probes, thought we would recommend

Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 570 nm.

Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 605 nm.

Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 650 nm.

Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 711 nm.
We'd also recommend multiple controls (although if signals are truly spatially exclusive, controls built in).

Any recommendations which of these is best? Are they all good? Any feedback appreciated. (But not the question, what are you doing at work between Christmas and New Year?)

Cheers-

Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
C: 914-309-3270 [hidden email]<mailto:[hidden email]> http://nyulmc.org/micros http://microscopynotes.com/

------------------------------------------------------------
This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
=================================

George McNamara

Re: additional probes to standard lineup, Brilliant Violet?

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Hi Michael,

Key: when labeling with Brilliant's or ThermoFisher's equivalent
SuperBrights, it is importnat to use the vendor's staining buffer (ships
in the product) because two or more will aggregate (of course any
antibody might aggregate anyway: I recommend spinning down Abs with a
chilled microfuge rotor and carefully aspirating and ussing the
supernatant).

I have only used BV421, loved it (I used it before the tandems were
available). I've written about Brilliant's in the context of
multiplexing with high resolution (the BV421 donor channel always has
some emission in the tandems), at

https://www.linkedin.com/pulse/resolution-blues-meets-21plex-salute-fluorescence-basic-mcnamara/

I also note that you can directly excite the tandem acceptors.

***

A couple of cool references I came across:

* Garry Nolan et al CODEX 30plex preprint
https://www.biorxiv.org/content/early/2017/10/20/203166 and
https://www.akoyabio.com/technology

* Pleiner et al secondary nanobodies doi: 10.1083/jcb.201709115
(http://jcb.rupress.org/content/early/2017/12/19/jcb.201709115) and
http://nano-tag.com (X4 anti-mouse not yet on their web site - learned
of it visiting their booth at ASCB-EMBO 2017).

I do not have any commercial interest in any of this post's content.
Hopefully BD or ThermoFisher will create, optimize, and sell, a panel(s)
of Brilliant's or SuperBright's secondary nanobodies so we can eliminate
the zoo of conventional immunofluorescence experiments and eliminate
supporting companies immunizxing and killing rabbits, goats, etc, for
our and our user's experiments.

Happy holidays,

George

p.s. today is a JHU holiday - my confocal feed comes to my home email.

On 12/29/2017 11:09 AM, Cammer, Michael wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> One of our clients wants to add a fifth or sixth probe to the standard Dapi, AF488, AF568, AF647 lineup.
>
> One of our confocals has a 594 nm laser and we also have true spectral detection, but rather than sandwich in overlapping probes, thought we would recommend
>
> Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 570 nm.
>
> Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 605 nm.
>
> Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 650 nm.
>
> Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at 407 nm and Emmax at 711 nm.
> We'd also recommend multiple controls (although if signals are truly spatially exclusive, controls built in).
>
> Any recommendations which of these is best? Are they all good? Any feedback appreciated. (But not the question, what are you doing at work between Christmas and New Year?)
>
> Cheers-
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
> C: 914-309-3270 [hidden email]<mailto:[hidden email]> http://nyulmc.org/micros http://microscopynotes.com/
>
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email.
> =================================

--

George McNamara, PhD
Baltimore, MD 21231
[hidden email]
https://www.linkedin.com/in/georgemcnamara
https://works.bepress.com/gmcnamara/75 (may need to use Microsoft Edge or Firefox, rather than Google Chrome)
http://www.ncbi.nlm.nih.gov/myncbi/browse/collection/44962650
http://confocal.jhu.edu

July 2017 Current Protocols article, open access:
UNIT 4.4 Microscopy and Image Analysis
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/abstract
supporting materials direct link is
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/full#hg0404-sec-0023
figures at
http://onlinelibrary.wiley.com/doi/10.1002/cphg.42/figures

Jeffrey Carmichael

Re: additional probes to standard lineup, Brilliant Violet?

In reply to this post by mcammer

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

*****COMMERCIAL RESPONSE*****

Hi Michael,

Like George, I also have no commercial interest in BD Biosciences and also
recommend BV421 as well as BV480. The link below does refer to Chroma's
optimized filter sets, hence the "COMMERCIAL" notice....

If workers can free up the channel normally used by DAPI by using one of
the far red DNA probes, that opens up two channels: both BV421 & BV480.
Even without giving up DAPI, one can fit in the BV480. Obviously, the
caveat is that all of these filters sets should be more narrow in bandwidth
than the more conventional filter sets for things like GFP or AF 488,
AF546, etc.

BV480 is very bright whereas DEAC is useless. Both of these BV dyes (and
most others) have exceptionally large extinction coefficients. It is
adequately separable from AF488, unlike CFP and related FPs.

If workers are using DAPI, then the cells are assumed to be dead (fixed) so
that allows for UV excitation, and whole breadth of the BUV tandems from
BD. These excite with a 350-375nm source and are visible as various
tandems all the way to 800nm. The native BUV395 is just that - it emits at
approx. 395nm, which is also unique.

George provided some very useful info, here's a link to an admittedly
"lightweight" but very illustrative example of a simple 5 color scheme for
the non-specialist which gives you the gist of how this could be expanded
upon by more ambitious workers:
https://www.chroma.com/5-channel-fluorescence-imaging-simplified

<https://www.chroma.com/5-channel-fluorescence-imaging-simplified>
Slides #7, 12 & 16 lay out possible labeling schemes with accompanying
filter recommendations. I've personally seen the recommended 5 colors/set
combinations on samples and was impressed by the ability to separate BV480
from BV488. The rest are a bit less challenging.

Finally, I think the best thing about these BV dyes is that they're bright
enough to use as directly conjugated primary antibodies.....if one can
conquer the chemistry. BD has been very helpful with some of my customers
with custom probes, but it's not easy.

Happy staining,
Jeff

*Jeff Carmichael*

*Technical and Product Marketing Manager**[hidden email]
<[hidden email]>*

On Fri, Dec 29, 2017 at 11:09 AM, Cammer, Michael <
[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> One of our clients wants to add a fifth or sixth probe to the standard
> Dapi, AF488, AF568, AF647 lineup.
>
> One of our confocals has a 594 nm laser and we also have true spectral
> detection, but rather than sandwich in overlapping probes, thought we would
> recommend
>
> Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at 407
> nm and Emmax at 570 nm.
>
> Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at 407
> nm and Emmax at 605 nm.
>
> Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at 407
> nm and Emmax at 650 nm.
>
> Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at 407
> nm and Emmax at 711 nm.
> We'd also recommend multiple controls (although if signals are truly
> spatially exclusive, controls built in).
>
> Any recommendations which of these is best? Are they all good? Any
> feedback appreciated. (But not the question, what are you doing at work
> between Christmas and New Year?)
>
> Cheers-
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
> NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> C: 914-309-3270 [hidden email]<mailto:[hidden email].
> edu> http://nyulmc.org/micros http://microscopynotes.com/
>
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of the
> intended recipient(s) and may contain information that is proprietary,
> confidential, and exempt from disclosure under applicable law. Any
> unauthorized review, use, disclosure, or distribution is prohibited. If you
> have received this email in error please notify the sender by return email
> and delete the original message. Please note, the recipient should check
> this email and any attachments for the presence of viruses. The
> organization accepts no liability for any damage caused by any virus
> transmitted by this email.
> =================================
>

--

<https://www.chroma.com/> CHROMA TECHNOLOGY CORP®
*an employee owned company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 | 802-428-2525
www.chroma.com | [hidden email]

Carol J. Bayles

Re: additional probes to standard lineup, Brilliant Violet?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

A little late, but check out the interesting chemistry behind the Brilliant Violet dyes:

http://www.bdbiosciences.com/us/applications/multicolor/s/brilliantdyes
http://www.sirigen.com/sirigen_technology.html

Carol
<><><><><><><><>
Carol Bayles
BRC-Imaging-FACS
B46 Weill Hall
Cornell University
Ithaca, NY 14853
607-275-9090 cell

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeffrey Carmichael
Sent: Friday, December 29, 2017 1:00 PM
To: [hidden email]
Subject: Re: additional probes to standard lineup, Brilliant Violet?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

*****COMMERCIAL RESPONSE*****

Hi Michael,

Like George, I also have no commercial interest in BD Biosciences and also recommend BV421 as well as BV480. The link below does refer to Chroma's optimized filter sets, hence the "COMMERCIAL" notice....

If workers can free up the channel normally used by DAPI by using one of the far red DNA probes, that opens up two channels: both BV421 & BV480.
Even without giving up DAPI, one can fit in the BV480. Obviously, the caveat is that all of these filters sets should be more narrow in bandwidth than the more conventional filter sets for things like GFP or AF 488, AF546, etc.

BV480 is very bright whereas DEAC is useless. Both of these BV dyes (and most others) have exceptionally large extinction coefficients. It is adequately separable from AF488, unlike CFP and related FPs.

If workers are using DAPI, then the cells are assumed to be dead (fixed) so that allows for UV excitation, and whole breadth of the BUV tandems from BD. These excite with a 350-375nm source and are visible as various tandems all the way to 800nm. The native BUV395 is just that - it emits at approx. 395nm, which is also unique.

George provided some very useful info, here's a link to an admittedly "lightweight" but very illustrative example of a simple 5 color scheme for the non-specialist which gives you the gist of how this could be expanded upon by more ambitious workers:
https://www.chroma.com/5-channel-fluorescence-imaging-simplified

<https://www.chroma.com/5-channel-fluorescence-imaging-simplified>
Slides #7, 12 & 16 lay out possible labeling schemes with accompanying filter recommendations. I've personally seen the recommended 5 colors/set combinations on samples and was impressed by the ability to separate BV480 from BV488. The rest are a bit less challenging.

Finally, I think the best thing about these BV dyes is that they're bright enough to use as directly conjugated primary antibodies.....if one can conquer the chemistry. BD has been very helpful with some of my customers with custom probes, but it's not easy.

Happy staining,
Jeff

*Jeff Carmichael*

*Technical and Product Marketing Manager**[hidden email]
<[hidden email]>*

On Fri, Dec 29, 2017 at 11:09 AM, Cammer, Michael < [hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> One of our clients wants to add a fifth or sixth probe to the standard
> Dapi, AF488, AF568, AF647 lineup.
>
> One of our confocals has a 594 nm laser and we also have true spectral
> detection, but rather than sandwich in overlapping probes, thought we
> would recommend
>
> Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 570 nm.
>
> Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 605 nm.
>
> Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 650 nm.
>
> Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 711 nm.
> We'd also recommend multiple controls (although if signals are truly
> spatially exclusive, controls built in).
>
> Any recommendations which of these is best? Are they all good? Any
> feedback appreciated. (But not the question, what are you doing at work
> between Christmas and New Year?)
>
> Cheers-
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> C: 914-309-3270 [hidden email]<mailto:[hidden email].
> edu> http://nyulmc.org/micros http://microscopynotes.com/
>
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of
> the intended recipient(s) and may contain information that is
> proprietary, confidential, and exempt from disclosure under applicable
> law. Any unauthorized review, use, disclosure, or distribution is
> prohibited. If you have received this email in error please notify the
> sender by return email and delete the original message. Please note,
> the recipient should check this email and any attachments for the
> presence of viruses. The organization accepts no liability for any
> damage caused by any virus transmitted by this email.
> =================================
>

--

<https://www.chroma.com/> CHROMA TECHNOLOGY CORP® *an employee owned company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 | 802-428-2525
www.chroma.com | [hidden email]

WAINWRIGHT James

Re: -|EXT|- Re: additional probes to standard lineup, Brilliant Violet?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

Does the polymer chain affect cell biochemistry? It looks pretty big.

I always understood it to be beneficial if the antibody / fluorophore was as small as possible to avoid influencing molecular behaviour.

James

James Wainwright
Global Applications Specialist - Microscopy Systems

Tel: +44 (0) 2890 237 126 ext. 2130
Skype: andor.j.wainwright
Mob: +44 (0) 7834 710 834
Web: https://www.andor.com/microscopy-systems

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carol J. Bayles
Sent: 10 January 2018 18:09
To: [hidden email]
Subject: -|EXT|- Re: additional probes to standard lineup, Brilliant Violet?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

A little late, but check out the interesting chemistry behind the Brilliant Violet dyes:

http://www.bdbiosciences.com/us/applications/multicolor/s/brilliantdyes
http://www.sirigen.com/sirigen_technology.html

Carol
<><><><><><><><>
Carol Bayles
BRC-Imaging-FACS
B46 Weill Hall
Cornell University
Ithaca, NY 14853
607-275-9090 cell

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeffrey Carmichael
Sent: Friday, December 29, 2017 1:00 PM
To: [hidden email]
Subject: Re: additional probes to standard lineup, Brilliant Violet?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

*****COMMERCIAL RESPONSE*****

Hi Michael,

Like George, I also have no commercial interest in BD Biosciences and also recommend BV421 as well as BV480. The link below does refer to Chroma's optimized filter sets, hence the "COMMERCIAL" notice....

If workers can free up the channel normally used by DAPI by using one of the far red DNA probes, that opens up two channels: both BV421 & BV480.
Even without giving up DAPI, one can fit in the BV480. Obviously, the caveat is that all of these filters sets should be more narrow in bandwidth than the more conventional filter sets for things like GFP or AF 488, AF546, etc.

BV480 is very bright whereas DEAC is useless. Both of these BV dyes (and most others) have exceptionally large extinction coefficients. It is adequately separable from AF488, unlike CFP and related FPs.

If workers are using DAPI, then the cells are assumed to be dead (fixed) so that allows for UV excitation, and whole breadth of the BUV tandems from BD. These excite with a 350-375nm source and are visible as various tandems all the way to 800nm. The native BUV395 is just that - it emits at approx. 395nm, which is also unique.

George provided some very useful info, here's a link to an admittedly "lightweight" but very illustrative example of a simple 5 color scheme for the non-specialist which gives you the gist of how this could be expanded upon by more ambitious workers:
https://www.chroma.com/5-channel-fluorescence-imaging-simplified

<https://www.chroma.com/5-channel-fluorescence-imaging-simplified>
Slides #7, 12 & 16 lay out possible labeling schemes with accompanying filter recommendations. I've personally seen the recommended 5 colors/set combinations on samples and was impressed by the ability to separate BV480 from BV488. The rest are a bit less challenging.

Finally, I think the best thing about these BV dyes is that they're bright enough to use as directly conjugated primary antibodies.....if one can conquer the chemistry. BD has been very helpful with some of my customers with custom probes, but it's not easy.

Happy staining,
Jeff

*Jeff Carmichael*

*Technical and Product Marketing Manager**[hidden email]
<[hidden email]>*

On Fri, Dec 29, 2017 at 11:09 AM, Cammer, Michael < [hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> One of our clients wants to add a fifth or sixth probe to the standard
> Dapi, AF488, AF568, AF647 lineup.
>
> One of our confocals has a 594 nm laser and we also have true spectral
> detection, but rather than sandwich in overlapping probes, thought we
> would recommend
>
> Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 570 nm.
>
> Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 605 nm.
>
> Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 650 nm.
>
> Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at
> 407 nm and Emmax at 711 nm.
> We'd also recommend multiple controls (although if signals are truly
> spatially exclusive, controls built in).
>
> Any recommendations which of these is best? Are they all good? Any
> feedback appreciated. (But not the question, what are you doing at work
> between Christmas and New Year?)
>
> Cheers-
>
>
> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
> 10016
> C: 914-309-3270 [hidden email]<mailto:[hidden email].
> edu> http://nyulmc.org/micros http://microscopynotes.com/
>
>
>
> ------------------------------------------------------------
> This email message, including any attachments, is for the sole use of
> the intended recipient(s) and may contain information that is
> proprietary, confidential, and exempt from disclosure under applicable
> law. Any unauthorized review, use, disclosure, or distribution is
> prohibited. If you have received this email in error please notify the
> sender by return email and delete the original message. Please note,
> the recipient should check this email and any attachments for the
> presence of viruses. The organization accepts no liability for any
> damage caused by any virus transmitted by this email.
> =================================
>

--

<https://www.chroma.com/> CHROMA TECHNOLOGY CORP® *an employee owned company*
10 Imtec Lane, Bellows Falls, Vermont 05101 USA
800-824-7662 | 802-428-2525
www.chroma.com | [hidden email]

___________________________________________________________________________This e-mail is confidential and is for the addressee only. Please refer to www.oxinst.com/email-statement for regulatory information.

George McNamara

Re: -|EXT|- Re: additional probes to standard lineup, Brilliant Violet?

*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
Post images on http://www.imgur.com and include the link in your posting.
*****

BV421 is 60 kDa, so small compared to an IgG molecule.

I suggest an even better approach will be BV421 site specifically
conjugated to a nanobody ~12 kDa (~110aa). See

Pleiner et al,

A toolbox of anti–mouse and anti–rabbit IgG secondary nanobodies

http://jcb.rupress.org/content/early/2017/12/19/jcb.201709115

DOI:10.1083/jcb.201709115

The company the above was licensed to is NanoTag Biotechnologies,
http://nano-tag.com/products which should have X4 anti-mouse and
anti-rabbit on their web page soon -- test samples I requested are in
transit to Baltimore right now. X4 = four nanobodies can bind each IgG
(two to light, two to heavy chain).

A couple of other companies in the fluorescent nanobodies field are:
* Chromotek
* Rockland Immunochemicals (including generating nanobodies from
immunized animals).

Anyone can search online for more companies that sell nanobody phage
display libraries. Ideally once hits are confirmed, affinity optimized,
(re)formatted, etc, the sequence(s) should be published (as Pleiner did)
to enhance reproducibility.

In 2018 the ethics of using irreproducible animal products is suspect,
doubly so when buying reagents from 'Santa Crap', which has been fined
by USDA for multiple reasons.

Quick pivot to another topic ... I encourage everyone to read Garry
Nolan's lab (and company) CODEX preprint, 30plex antibody detection
https://www.biorxiv.org/content/early/2017/10/20/203166

which I think would be even more efficient if 'tagging' the primary
antibodies was replaced with 'tagged' X4 nanobodies.

Sincerely,

George
p.s. no financial interest in any companies mentioned here. Nor do I
have a short position on Santa Crap.

On 1/11/2018 3:26 AM, WAINWRIGHT James wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Does the polymer chain affect cell biochemistry? It looks pretty big.
>
> I always understood it to be beneficial if the antibody / fluorophore was as small as possible to avoid influencing molecular behaviour.
>
> James
>
> James Wainwright
> Global Applications Specialist - Microscopy Systems
>
> Tel: +44 (0) 2890 237 126 ext. 2130
> Skype: andor.j.wainwright
> Mob: +44 (0) 7834 710 834
> Web: https://www.andor.com/microscopy-systems
>
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Carol J. Bayles
> Sent: 10 January 2018 18:09
> To: [hidden email]
> Subject: -|EXT|- Re: additional probes to standard lineup, Brilliant Violet?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> A little late, but check out the interesting chemistry behind the Brilliant Violet dyes:
>
> http://www.bdbiosciences.com/us/applications/multicolor/s/brilliantdyes
> http://www.sirigen.com/sirigen_technology.html
>
> Carol
> <><><><><><><><>
> Carol Bayles
> BRC-Imaging-FACS
> B46 Weill Hall
> Cornell University
> Ithaca, NY 14853
> 607-275-9090 cell
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jeffrey Carmichael
> Sent: Friday, December 29, 2017 1:00 PM
> To: [hidden email]
> Subject: Re: additional probes to standard lineup, Brilliant Violet?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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> *****
>
> *****COMMERCIAL RESPONSE*****
>
> Hi Michael,
>
> Like George, I also have no commercial interest in BD Biosciences and also recommend BV421 as well as BV480. The link below does refer to Chroma's optimized filter sets, hence the "COMMERCIAL" notice....
>
> If workers can free up the channel normally used by DAPI by using one of the far red DNA probes, that opens up two channels: both BV421 & BV480.
> Even without giving up DAPI, one can fit in the BV480. Obviously, the caveat is that all of these filters sets should be more narrow in bandwidth than the more conventional filter sets for things like GFP or AF 488, AF546, etc.
>
> BV480 is very bright whereas DEAC is useless. Both of these BV dyes (and most others) have exceptionally large extinction coefficients. It is adequately separable from AF488, unlike CFP and related FPs.
>
> If workers are using DAPI, then the cells are assumed to be dead (fixed) so that allows for UV excitation, and whole breadth of the BUV tandems from BD. These excite with a 350-375nm source and are visible as various tandems all the way to 800nm. The native BUV395 is just that - it emits at approx. 395nm, which is also unique.
>
> George provided some very useful info, here's a link to an admittedly "lightweight" but very illustrative example of a simple 5 color scheme for the non-specialist which gives you the gist of how this could be expanded upon by more ambitious workers:
> https://www.chroma.com/5-channel-fluorescence-imaging-simplified
>
> <https://www.chroma.com/5-channel-fluorescence-imaging-simplified>
> Slides #7, 12 & 16 lay out possible labeling schemes with accompanying filter recommendations. I've personally seen the recommended 5 colors/set combinations on samples and was impressed by the ability to separate BV480 from BV488. The rest are a bit less challenging.
>
> Finally, I think the best thing about these BV dyes is that they're bright enough to use as directly conjugated primary antibodies.....if one can conquer the chemistry. BD has been very helpful with some of my customers with custom probes, but it's not easy.
>
> Happy staining,
> Jeff
>
>
> *Jeff Carmichael*
>
> *Technical and Product Marketing Manager**[hidden email]
> <[hidden email]>*
>
> On Fri, Dec 29, 2017 at 11:09 AM, Cammer, Michael < [hidden email]> wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> Post images on http://www.imgur.com and include the link in your posting.
>> *****
>>
>> One of our clients wants to add a fifth or sixth probe to the standard
>> Dapi, AF488, AF568, AF647 lineup.
>>
>> One of our confocals has a 594 nm laser and we also have true spectral
>> detection, but rather than sandwich in overlapping probes, thought we
>> would recommend
>>
>> Brilliant Violet 570(tm) is a fluorescent polymer tandem with Abmax at
>> 407 nm and Emmax at 570 nm.
>>
>> Brilliant Violet 605(tm) is a fluorescent polymer tandem with Abmax at
>> 407 nm and Emmax at 605 nm.
>>
>> Brilliant Violet 650(tm) is a fluorescent polymer tandem with Abmax at
>> 407 nm and Emmax at 650 nm.
>>
>> Brilliant Violet 711(tm) is a fluorescent polymer tandem with Abmax at
>> 407 nm and Emmax at 711 nm.
>> We'd also recommend multiple controls (although if signals are truly
>> spatially exclusive, controls built in).
>>
>> Any recommendations which of these is best? Are they all good? Any
>> feedback appreciated. (But not the question, what are you doing at work
>> between Christmas and New Year?)
>>
>> Cheers-
>>
>>
>> Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory NYU
>> Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY
>> 10016
>> C: 914-309-3270 [hidden email]<mailto:[hidden email].
>> edu> http://nyulmc.org/micros http://microscopynotes.com/
>>
>>
>>
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