Re: THP-1's and fluorescent dyes

PAUL@flowcyt.cyto.purdue.edu
Tue, 17 Dec 1996 17:20:19

We have done many of these studies with a variety of cells including
macrophages, neutrophils, endothelial cells and chondrocytes.
Basically the dyes mentioned all work to a degree - you need to load
your particular cells, determine the loading time (collect histograms
every 5 minutes for 45 minutes (some cells such as endothelial cells
take 45 mintues, PMN only 10-15 mins). Stimulate the cells and
measure over time fro another 15-60 mins (again depending on cell).
The assay is one of the easier assays to do. You need lots of
controls to interpret your data such as an unstimulated but loaded
tube so that you knwo if the fluorescence is changing in teh absence
of stimulator - and this must be taken for every measurement you
take. Subtract this from your test specimen each time.

I don't suppose you can wait till April as CURRENT PROTOCOLS IN
CYTOMETRY is filled with wonderful methods including exactly what you
want! Happy Christmas!

Paul

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Subject: THP-1's and fluorescent dyes
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Has anyone worked with the THP-1 cell line using Dihydrorhodamine 123 (DHR)
or Hydroethidine (HE)? I am in search of a method to stimulate these cells
with LPS, an inflammatory cytokine, or virus, and measure oxidative burst.
As of yet, my efforts have come up empty, but I thought there may be some
unpublished or hard to find methods out there in Flowland. Any help,
insights or watch-outs will be appreciated.

Thanks...Jodi McKenzie-Kroeger
J.Paul Robinson, Purdue University Cytometry Labs
Professor of Immunopharmacology
robinson@flowcyt.cyto.purdue.edu PH:317-494 6449 FAX:317-494 0517
web http://www.cyto.purdue.edu

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CD-ROM Vol 3 was produced by Monica M. Shively and other staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone: (765)-494-0757; FAX(765) 494-0517; Web http://www.cyto.purdue.edu , EMAIL cdrom3@flowcyt.cyto.purdue.edu