mr_redram at hotmail.com
Fri Mar 10 09:36:05 EST 2000
My experience indicates that PF fixing of GFP cells significantly decreases
the fluorescence intensity of these cells.
>From: Candace Enockson <enockson at musc.edu>
>To: Cytometry Mailing List <cytometry at flowcyt.cyto.purdue.edu>
>Subject: RE: GFP
>Date: Thu, 09 Mar 2000 10:06:13 -0400
>I have done a fair amount of work on GFP transfected cells and have found
>them readily in the 530nm range, but these are unfixed cells. I wonder,
>too , if your fixing has something to do with loss of staining. As I
>recall there was some discussion about this topic not so long back on this
>website. You may want to go into the archives and look for it. Good luck.
>Medical Unversity of South Carolina
>--On Wed, Mar 8, 2000 3:16 PM +0100 Carl-Magnus Hogerkorp
><carl-magnus.hogerkorp at molmed.lu.se> wrote:
> > Dear all,
> > having worked with hematopoetic cells transduced with a GFP containing
> > vector, cells then retransplanted into mice, we have experienced
> > difficulties when analyzing MNCs from such mice. Not to bore you with
> > details, we eventually found out that a lysis kit we used from BD
> > negatively affected the GFP expression to a very high extent compared to
> > samples treated with ordinary NH4Cl lysis. Although I do not know the
> > details on how GFP is affected with different fixation procedures, the
> > lysis kit we used contained formaldehyde. As I see it, this could effect
> > the GFP in the cells either causing it to leak out from the cells, or
> > somehow interacting on the protein to reduce the emission. If it is the
> > latter, perhaps you could test not to treat your sample with
> > and compare how it looks with such a fixation procedure.
> > David Bryder
> > Stem Cell Laboratory
> > Lund, Sweden
> > David.Bryder at molmed.lu.se
> > -----Original Message-----
> > From: Slava Epelman [mailto:sepelman at ucalgary.ca]
> > Sent: den 7 mars 2000 01:59
> > To: Cytometry Mailing List
> > 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 at ucalgary.ca
> > University of Calgary
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