Any chance it is being secreted? Check some culture supernatant in a
fluorometer maybe?
David McFarland
Howard Hughes Medical Institute
Flow Cytometry Facility
Vanderbilt University Medical Center
---------------------- Forwarded by David McFarland/VUMC/Vanderbilt on
03/07/2000 02:43 PM ---------------------------
Slava Epelman <sepelman@ucalgary.ca> on 03/06/2000 06:59:14 PM
(Embedded image moved to file: pic09469.pcx)From:(Embedded image moved to
file: pic14065.pcx)Slava Epelman <sepelman@ucalgary.ca> on 03/06/2000 06:59 PM
To: Cytometry Mailing List
<cytometry@flowcyt.cyto.purdue.edu>
cc: (bcc: David McFarland/VUMC/Vanderbilt)
Subject: Re: GFP
To anyone who has worked with GFP, we need some help.
The protein of interest in our lab is able to stimulate monocytes
(increased cytokine production) and T-cells (up-regulation of CD69). We
are currently trying to determine which population of PBMC it interacts
with, if it has saturateable receptors, and eventually whether it is
internalized. We have linked the protein to GFP (mutant 3), which is a
FACS optimized mutant of wild-type GFP and is read in the FL-1 channel.
The GFP link does not modify the function of our protein, but we have
not been able to detect it on PBMC or a responsive monocytic cell line
(THP-1). Our FACS machine has an argon laser and excites at 488 nm. And
I believe it reads emissions in the FL-1 channel between 515-545 nm. We
have done the following to try and detect GFP on any cell population,
without any success.
1. Taken PBMC and THP-1 cells, and stimulated in culture with varying
doses of the GFP tagged protein for 15-30 min at 37C, washed off the
media in FACS wash (PBS, 1% FCS, 0.1% NaN3) and fixed in 1% buffered
formalin (stock solution)
2. Taken THP-1 fixed in 1% buffered formalin, washed off the formalin in
FACS wash , incubated various concentrations of the GFP tagged protein
for 30 min, washed it off with FACS wash and resuspended in 1% buffered
formalin
3. Taken PBMC and THP-1 cells, washed with FACS wash, incubated with
GFP-protein for 30 min at 4C, washed off unbound protein and fixed in 1%
buffered formalin.
We used positive controls such as anti-CD4, anti CD64 and anti
CD14-FITC, and detected a strong signal for each, although no signal was
detected for our GFP-protein.
On a fluorimeter we measured the how "green" our protein was. It was 10
fold less intense than IgG-FITC (when equalized for mass) that we
purchased from BD, when excited at 485 nm and the emission read at 535
nm.
We also thought of using the fluorimeter to measure GFP bound cells with
the idea being that if the receptor is present in a low amount, then
increased number of cells may increase the total GFP bound. However the
fluorimeter, despite being quite new, was not nearly as sensitive as our
FACS machine. Loading of 2x10^6 cells/well still did not show a
detectable signal, and signals for anti-CD14, Cd64 and CD4-FITC were
only about 1.5-3 times greater than the background.
We have though of trying to biotinylate the protein and then use
PE-conjugate steptavidin, or perhaps FITC labeling the protein itself.
We have also contemplated linking the GFP-protein to beads, and thereby
increasing the signal of a binding event, but that may prevent any
future studies looking at whether the protein is internalized, as it may
be the bead that is inducing internalization.
Any help would be greatly appreciated,
Slava Epelman
sepelman@ucalgary.ca
University of Calgary
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