Fixation/Permeabilization for intracellular antigen, BrdU detection, and surface staining

C. Kevin Becker ckb at phnxflow.com
Mon Apr 20 00:46:48 EST 1998


Ed,

Phoenix Flow Systems manufactures and sells a kit for measuring BrdU
incorporation simultaneously with intracellular antigens and surface
staining.  Our kit (called Absolute-S) uses light to make breaks in the DNA
where ever the Br has incorporated into the DNA during the BrdU labeling
part of your experiment.  No acid or heat denaturation is required to unwind
the DNA to get the anti-BrdU antibody in.  This methodology is called Strand
Breaks Induced by Photolysis (SBIP) and was discovered by Darzynkiewicz and
Traganos.  Please see our web page at www.phnxflow.com which has a complete
explanation of the protocol of this assay.

Dr. Danzynkiewicz will be speaking at the flow users meeting on May 1 at
your university, UC Davis.  I will also be at the meeting.

Sincerely,

Kevin
   ______   ______  _______
/_____/  /_____   /______   C. Kevin Becker
/            /             ______/ Phoenix Flow Systems, Inc.
11575 Sorrento Vlly. Rd. #208, San Diego, CA 92121 USA
(619) 453-5095 FAX (619) 259-5268 ckb at phnxflow.com


>>From: Ed Fulton <befulton at UCDAVIS.EDU>
>>X-Sender: ez043830 at catbert.ucdavis.edu
>>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>>Subject: Fixation/Permeabilization for intracellular antigen, BrdU
>detection, and surface staining
>>
>>
>>Dear Flowers,
>>
>>I'm going to be trying to simultaneously detect BrdU incorporation, the
>>presence of an intracellular antigen (the capsid protein of bovine
>>leukemia virus), and surface lineage markers (such as IgM).  I've been
>>able to detect surface IgM and capsid simultaneously on Flow using
>>Caltag's Fix and Perm.  Previously, I've looked at capsid and BrdU
>>simultaneously on slides using a hot formamide treatment.  Before I start
>>the process of trial and error and use expensive reagents and machine
>>time, I was wondering if someone could suggest methods that have worked
>>for them that allow simultaneous measurement of BrdU incorporation and an
>>internal viral protein while maintaining enough cell integrity to allow
>>accurate surface lineage determination.  Samples are Peripheral Blood
>>Cells from Sheep or Sheep lymphocyte cell lines and machine currently
>>being used is a BD FACScan although switch to dual-laser BD sorter may be
>>in future.
>>
>>Thanks for any comments or hints,
>>
>>Ed Fulton
>>Graduate Student in Microbiology
>>U.C. Davis
>>
>> *********************************
>> * Ed Fulton *
>> * Animal Science, U.C. Davis *
>> * 1 Shields Avenue *
>> * Davis, CA 95616 *
>> * befulton at ucdavis.edu *
>> * lab# (530) 752-1492 *
>> * Home of D2 Champs - UCD Aggies*
>> *********************************
>>
>>
>>
>






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