We are not using an Enterprise; we use an I-70 at 200 mW of 488 nm
light. We did not test PE-Cy5 since it excites similar to APC with
the 633 nm line of the HeNe.
Marty
>Dear Marty,
>
>These figures you get, are they of the standard Coherent enterprise II and
>the Spectra Physics HeNe?
>What about PECy5?
>
>/Carl-Magnus
>
>Carl-Magnus Högerkorp
>Dept. of Immunotechnology
>University of Lund
>Box 7031
>22007 Lund
>Sweden
>
>Tel. +46-46-222 7622
>Fax. +46-46-222 4200
>
>-----Original Message-----
>From: Marty Bigos [mailto:mbigos@gladstone.ucsf.edu]
>Sent: den 11 augusti 2001 02:21
>To: Cytometry Mailing List
>Subject: Re: Phar Red supplementary question
>
>
>>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
>
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