[ Alexa selection question ]

Eric Van Buren eric.vanburen at wayne.edu
Fri Jan 5 18:01:53 EST 2007

Alamelu Raja,

No, you cannot detect Alexa Fluor 700 in FL3. According to the manufacturer's
website, www.invitrogen.com, Alexa Fluor 700 has <1% absorption at 488 nm.

However, they do (or perhaps Caltag does) offer a tandem conjugate of R-PE to
Alexa Fluor 700; this would work for FL3. So too would the PE-Cy5.5, PE-Cy7
PerCP-Cy5.5, PE-Alexa Fluor 680, and PE-Alexa Fluor 750 tandem conjugates,
as well as plain old PerCP. [This list may not be exhaustive.]

PE-Cy5 and PE-Alexa Fluor 647 should be avoided when using APC (or Cy5 or
Alexa Fluor 633 or Alexa Fluor 647) in FL4 of the Calibur. This is because
the Cy5 or Alexa Fluor 647 component of the PE tandem dyes can be directly
excited by the ~635 nm laser (and thus show up in FL4 equally as well as FL3).

Remember, on the Calibur FL4 is ~635 nm excitation with a 660/10 band pass
filter (centered at 660 nm with a 10 nm width, i.e., cut-on at 655 nm and
cut-off at 665 nm). FL3 is 488 nm excitation with a 670 nm long pass filter.
(The Scan, and Caliburs without the FL4 option, use a 650LP for FL3.)

Keep your Calibur clean and always run the time delay calibration prior
to running samples requiring FL4. If it fails, you may have a difficult
time setting compensation because you are not accurately measuring the
FL4 fluorescence. (Using software compensation doesn't cure the
problem.) If cleaning doesn't correct the failure, you may need a service
visit. Accidentally bumping the diode laser is certain to require service
unless you can figure out how to align it yourself, so don't touch it!
Changes in the flow rate -- sometimes just the result of normal part
age/wear -- can also cause the time delay calibration to fail. Regularly
scheduled preventive maintenance should prevent these failures. The Scan
is far less picky in this regard (because it only has one laser), and
newer BD instruments (e.g. LSR II) have a better "time delay calibration"
user interface. So treat your Calibur with extra care!

Best of luck,

>Dear Flow list members,     We have a FACS Calibur with 2 lasers - 488 and ~635nm and
emission optics to measure FL1, FL2, FL3 and FL4.     Can we use Alexa Flour 700 as FL3?
When viewing the Spectrum at BD site, Alexa Fluor is falling totally within the range of
FL3 emission filter without any overlap with FL2 or FL4.     But will the 635 laser be
able to excite Alexa700?	   Will Alexa 647 be useful, instead of Alexa700 and
without interference with FL4 APC?     Will PE-Cy5 be useful, instead of Alexa700 and
without interference with FL4 APC?     Those who have experience with FACS Caliber,
kindly give your suggestions please.	 With thanks,  Alamelu Raja   
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Eric Van Buren <eric.vanburen at wayne.edu>
Manager, Flow Cytometry Core Facility
Karmanos Cancer Institute
Detroit, Michigan, USA

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