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
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