Hi Len,
The four colour combination I use with those lasers on my Vantage is:
Fluorescein, PE, PE-Texas Red (eg Red613 or Coulter's ECD), and APC
for FL1, Fl2, Fl3 and FL4 respectively. The PE-TR emission is picked
up in FL3 using a 610 nm filter, which avoids the 488-excited APC
fluorescence, and doesn't spill over appreciably into FL4 (unlike
PE-Cy5 for instance which excites in both lasers). We're lucky in
that we can use fl2,3and 4 to define CD8,CD45RO and CD28 and then
vary the FL1 reagent for a range of Vbeta usage - it might be
trickier to meet your particular needs with the available range of
ECD and APC conjugated antibodies.
You might need to use extreme compensation levels between FL2 and FL3
depending on PE-TR conjugate and filter setup (try using a 575 DF20
rather than a 585 DF30 for FL2), but balancing the dichroics seems to
work quite nicely for me.
If you just want to analyse you could use a wider range of
combinations and compensate off-line using FCSPress, FlowJo or
WinList (and possibly other packages?) which will allow you to apply
the necessary cross-beam compensation.
The chances are you'll want to sort some of those populations
eventually and feel more comfortable compensating live - so you'll
need to trade off between reagent panels that minimise cross-over and
hardware that will remove it, like OmniComp and the "Diva".
Good luck
Ray
ps I haven't compared CD11c reagents
>Dear All
>
>We are attempting to set up 4 colour assays on our FACS Vantage (NOT SE
>upgraded) using APC on a HeNe laser delivering about 30 mW. Abs are from BD.
>
>1) Should we expect to see the APC signal in the FL3 channel (optimised for
>PECY5) of our 488 nm line? ie. Is APC excited at all by the 488 nm laser?
>
>2) Is anybody using BD's CD11c-APC mAb on a similar system, if so how does
>the signal compare with PE.
>
>Any help or hints on setting up 4 colour on a Vantage with 488 and 635 nm
>laser lines would be most appreciated.
>
>Len Brown
--
Ray Hicks
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