My experience indicates that PF fixing of GFP cells significantly decreases the fluorescence intensity of these cells. Phil Marder >From: Candace Enockson <enockson@musc.edu> >To: Cytometry Mailing List <cytometry@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. >Candace Enockson >Medical Unversity of South Carolina > >--On Wed, Mar 8, 2000 3:16 PM +0100 Carl-Magnus Hogerkorp ><carl-magnus.hogerkorp@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 >technical > > difficulties when analyzing MNCs from such mice. Not to bore you with >the > > 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 >BD > > 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 >formaldehyde > > and compare how it looks with such a fixation procedure. > > > > > > David Bryder > > Stem Cell Laboratory > > Lund, Sweden > > > > David.Bryder@molmed.lu.se > > > > -----Original Message----- > > From: Slava Epelman [mailto:sepelman@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@ucalgary.ca > > University of Calgary > > > > > > > > ______________________________________________________ Get Your Private, Free Email at http://www.hotmail.com
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