>Hi Nicole Et Al
>Can you please clarify "high" vs "low" laser power in this context?
>
>I have one of those OLD BD/Spectra Physics HeNe lasers that you
>can't kill with a big stick, still giving about 45mW after all these
>years...
Hi Joseph -
We have been using a 35 mW HeNe to excite APC and Cy7APC for 6 color
panels of reagents here. The laser works quite well for APC. It
works OK for Cy7APC as a single color stain, but it is the poorest of
our 6. The table below shows the ratio of positive to negative
populations of CD8 stained human pbmc's, normalized to FITC, run on a
Vantage SE:
color ratio
FITC 1.00
PE 5.37
PE-TR 1.69
PE-Cy7 1.79
APC 10.27
APC-Cy7 0.34
Your milage may vary, but I would expect your HeNe to do at least as
well as this. It is critical, though, to have appropriate filters and
red-sensitive PMTs on both APC and Cy7APC channels.
The big problem, as Nicole pointed out, is that the absolute light
level of Cy7APC is low, and it has emission in the APC channel, due
to incomplete energy transfer to the Cy7 fluorophore. So when you
compensate between the two colors, bright Cy7APC staining will have a
compensated background in the APC channel significantly higher than
the APC signal of unstained cells. This will degrade your ability to
separate low, and even moderately bright APC signals from background.
As Nicole suggested, one way to live with this is to select a reagent
for Cy7APC which is bright enough to show up on that channel, but no
brighter, so the background on APC will have the least increase, and
to put a bright reagent on APC that it will be above the increased
background caused by the Cy7APC.
Good Luck!
Marty
>I was hoping to use that for APC & APC-Cy7 soon; will it do, or do
>we need 100mW that won't last?
>
>Joseph.
>
>At 11:30 9/8/2001, Nicole Baumgarth wrote:
>{in relation to another question...}
>
>>As for the general "disappointing" staining with Cy7APC.....can you
>>increase laser power? All of the tandem-dyes that I have tried seem to be
>>doing increadibly much better at higher laser power. The more the better it
>>seems. This is really a limitation for use of the dyes when you try to use
>>them with low-power lasers.
--
Marty Bigos
Director
The J. David Gladtone Institutes Flow Cytometry Core
mbigos@gladstone.ucsf.edu
(voice) 415-695-3832
(fax) 415-826-8449
Mailing Address:
Gladstone Institutes
P.O. Box 419100
San Francisco, CA 94141-9100
Shipping Address:
Gladstone Institutes
365 Vermont Street
San Francisco, CA 94103
FedEx, UPS, or courier:
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