>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.
I have a similar problem with APC-Cy7 as Andy does. Something could
be wrong with my optics, etc.; I am looking into that. However, there
are two other factors to consider:
1) Source of reagent - I have used various CD8 conjugations
and second step SA conjugates with widely varying results; from a
half decade above autofluorescence to about one and a half decades. I
have never seen on my instrument 2.5 decades above background.
2) Excitation - Both Andy and I use a HeNe, while I surmise
you are using a dye laser to excite this reagent. Other folks may be
using the 647 nmm krypton line. This also could make a difference and
should be investigated as well.
Marty
>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
--
Marty Bigos
Director, Flow Core
415-695-3832
Gladstone Institute of Virology and Immunology
Building 3 SFGH
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:57:59 EST