Re: Intracellular staining with QuantumDots

I have to disagree with this.  

Let me repeat: QDots are smaller than PE, so anything you can do with PE
conjugates is  theoretically possible with qdot conjugates. 

The issue of multiple abs per qdots is also the same issue with PE--since
the same chemistry is used to conjugate qdots to ab as is used to conjugate
PE to qdots, the conjugates will show similar size.  Note that even 2 Abs on
a single qdot will likely be smaller in physical size than one ab with one
PE... And most PE conjugates will have 2 PE's per Ab (not to mention
containing higher order complexes), which will make them bigger than
virtually any complex of Abs to qdot.

But let's also remember that physical size scales with the cube root of mol
mass (roughly).  So even if a conjugate were 8 times as massive as another,
it's size is only twice as "wide".

But, as always, the proof is in the pudding... And we have had no problems
getting qdot-conjugated antibodies into cells using standard fix/perm
conditions.  However, we have not done this enough for me to speak to
background binding, relatice avidity, etc.  But they get in just fine!

So, no microinjection, nothing complicated.  

mr

(And yes, I know qdots are more dense than proteins, and may have a slight
difference in diffusion kinetics.  However, the difference in density of a
qdot-ab conjugate is trivial compared to a pe-ab conjugate--not to mention
that diffusion kinetics is almost certainly irrelevant in IC staining.	But
even if it were relevant, it would be taken into account by the titration
that you would have done on the conjugates already!)

mr


-----Original Message-----
From: Raquel Ibanez <ribanez@els.mq.edu.au>
To: cyto-inbox
Roederer, Mario  (NIH/VRC) <Roederer@nih.gov>
Sent: Thu Mar 10 17:32:01 2005
Subject: RE: Intracellular staining with QuantumDots

Mario,

If I understand Max's experiments. He is trying to stain cells with FITC
conjugated to antibodies and Qdots conjugated to antibodies. In this case,
Qdots can not diffuse through the cells membranes using normal
permeabilization procedures because they are not that small. As you say,
Qdots are tiny crystals. However, when Qdots are conjugated to biotin,
streptavidin or any other chemical compound to hybridise the specific
antibody, then the size of the Qdots increase. Also, keep in mind that
normally one single Qdot can attach more than one antibody on its surface
what makes them even bigger. I reckon Max will have to do procedures
including microinjection, cationic lipid-based reagents or do conjugation to
membrane-permeable peptides.

On the other hand, if Max is using plain unlabeled Qdots, he will also need
to do more aggressive permeabilization procedures because Qdots are bigger
than simple FITC molecules (for example sonication). But int his case,	the
staining of the Qdots will be completely unspecific.

I think you can find more info at the Quantum dot corporation website

Cheers

Raquel

Raquel Ibáñez Peral
Australian Centre for Astrobiology
Dpt. of Earth and Planetary Science
Macquarie University
2109, Sydney, NSW, Australia
Tlf: 02- 9850 6285
ribanez@els.mq.edu.au


>>> Mario Roederer <roederer@nih.gov> 03/10/05 6:09 am >>>
I want to correct one common misconception about QDots:  they are NOT 
very large particles.  In fact, QDots are smaller than PE.  Thus, any 
fixation/permeabilization procedure you use for PE-conjugates should 
in principle work for QDots.

mr

At 4:42 PM -0800 3/7/05, Robert C. Leif wrote:
>    Since QDots are very large particles, you will have to permeabilize the
>cells. However, as a totally biased party, I suggest that you might try the
>europium Quantum Dye(R). The molecular weight of the monoisothiocyanate is
>674 daltons. The Quantum Dye molecular volume is approximately 0.45% that
of
>a Qdot. The europium Quantum Dye is excited at 365 nm and emits at 619 nm.
>The emission width at half maximum, 5.2 nm, is approximately one fourth
that
>of a Qdot. Because the lifetime of the europium Quantum Dye is
approximately
>one millisecond, it is presently unsuited for flow cytometry and fast laser
>scanning systems.  More information including abstracts and papers can be
>found at www.newportinstruments.com 
>	Bob Leif
>	rleif@rleif.com 
>	-----Original Message-----
>    From: Max Warncke [mailto:maxwarncke@ema-syn.de] 
>    Sent: Friday, March 04, 2005 1:47 PM
>    To: Cytometry Mailing List
>    Subject: Intracellular staining with QuantumDots
>	Do I have to use a very strong permeabilization protocol if I want
to
>stain
>	intracellular antigens with QDots?
>	I tried to stain MHCII in and on murine (not fully matured) DC and
>	visiualized them on the confocal. Compared to FITC I get a brighter
>	extracellular fluorescence with QDots (525), but with the FITC
>antibody I
>	can see a bright signal inside the cell, which is totally absent
with
>QDots.
>	For permeabilization I used Triton-X, counterstained with DAPI and
>	Phalloidin Alx594.
>	Many thanks
>	Max


-- 
_____________________________________________
Mario Roederer, Ph.D.
Chief, ImmunoTechnology Section and Flow Cytometry Core
Vaccine Research Center, NIAID, NIH
40 Convent Dr., Room 5509
Bethesda, MD 20892-3015
Phone: 301 594-8491
FAX: 301 480-2651