Andy,
You should check your PMTs and filters for the Cy7APC. This dye is
really quite bright; if you don't get at least as good a separation
for any given antibody as you would with a fluorescein conjugate of
that antibody, then there is a problem with your optics or your
conjugate. Has nothing to do with Vantage vs. MoFlo; I've done it on
both platforms with outstanding results. Despite potential claims to
the contrary, sensitivity is essentially equivalent on the two.
As I got your EMail, I was analyzing some compensation samples... our
Cy7APC-CD8 conjugate on human PBMC gives positive peak that is 2.5
decades above the autofluorescent cells. If you aren't at least 1.5
decades above autofluorescence with CD8 conjugates, then something is
wrong.
It may have something to do with the particular conjugate; you might
want to try some test conjugates from different manufacturers. In
addition, it is critical to have "red-sensitive" PMTs for Cy7
detection. Standard PMTs will work, but the red-sensitive will give
you up to an order of magnitude (realistically, 3-fold) more
sensitivity. Besides the PMTs, you need to be particularly aware of
the optical quality of each of your filters and dichroics. Make sure
you have the highest quality available ("AR
coating"--anti-reflectance coating improves signal 10-20%). Having
the right band passes can improve signal as much as 2-fold. Given
the number of optical elements you may have on your system,
optimizing each one can give you a significant boost in signal.
As for your UV dyes, you're out of luck. Cascade Blue will be
marginal (at best) on a UV line; in fact, it will only work for the
most highly-expressed epitopes (CD8, for example, is barely above
autofluroescence). You will not be able to detect Cascade Yellow or
Alexa 430 off of a UV excitation. To use these dyes, you will need a
405 nm line--currently costing $50K for a big Krypton laser--although
this cost will come down with new solid state 405 nm lasers (which
currently can't put out very much power, but appear to work well for
Cascade Blue). Given today's dyes, it is simply not feasible to do
much immunophenotyping with a UV laser. BTW, using a high power 405
line, we get more than 2 decades of signal for Cascade Blue CD8; less
than 1 decade for Alexa 430 CD8. Note that Cascade Yellow and
Cascade Blue have a low, "nonspecific" binding affinity for each
other; you have to be very careful when using conjugates of these two
simultaneously. Alexa 430 does not have this property.
You will also be able to (eventually) increase sensitivity if you
purchase the digital electronics option. The digitization combined
with peak area measurements improves signal detection over analog,
particularly when compensation is involved (which it always is). We
are in the process of determining how much of a gain this represents,
especially when combined with slowing down the jet velocity--see our
posters at CAC or ISAC. By the way, if you are running at high
pressure--don't! Unless you need the speed for sorting, the jet
velocity should be turned down as far as possible in order to get
best signal. The gain in signal will be best for area measurements,
however.
mr
At 10:33 AM -0700 10/22/01, Andy Johnson wrote:
>Dera Flowers
>
>How many people are using 6 colours on a Vantage ? 3 colours off the 488,
>APC plus APC-Cy7 (very weak) with the 633 and Hoechst is fine off the UV.
>Unfortunately I need colours such as cascade blue/yellow and something
>brighter than the APC-Cy7.
>
>Can anybody who uses more than the standard colours give me a suggestion as
>to how to either increase sensitivity or which colours work best. I also
>have a feeling that it maybe an issue between the Vantage and the MoFlo
>(being more sensitive). I know all the available fluorochromes out there,
>so what I need to know is which one people have used and got to work.
>
>Would a 120mW from a I-70 laser greatly increase the UV signals, or should
>the 60mW from an enterprise be sufficient ?
>
>Hoping that somebody can help ?
>
>Andy
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