Date: Fri Oct 14 2005 - 04:21:18 EST
Hello dear flowers, for those interested in LacZ detection by FACS using the FDG substrate I have listed the answers I have got so far. We didn't repeat the experiment yet with my collegue who has the Rosa mice but we are planning to use osmotic shock at 37 degrees for 30 min with FDG + Verapamil (to reduce the leaking of the FDG out of the cells) and Chloroquine (to reduce degradation of the FDG by the lysosomes) as this protocol seems to work the best for the loading of the cells rather than the MP kit. X-gal activity has been proven in our mice by histology. I will keep you updated when we will have the results. Greetings, Simona 1/Dear Simona, I suppose this excerpt from the Molecular Probes handbook will explain it: The fluorescent hydrolysis product of FDG is fluorescein (<http://www.invitrogen.com/content.cfm?pageid=8012&sku=F1300>F1300, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=F36915>F36915; <http://probes.invitrogen.com/handbook/sections/1001.html>Section 10.1; <http://probes.invitrogen.com/handbook/figures/1042.html>Figure 15.3), which rapidly leaks from cells under physiological conditions, making the use of FDG problematic for prolonged studies. Below you can see how fast the leakage is. It doesn’t matter if the substrate is FDG or FDA. 5c983930.jpg Figure 15.3 Loading and retention characteristics of intracellular marker dyes. Cells of a human lymphoid line (GePa) were loaded with the following cell-permeant acetoxymethyl ester (AM) or acetate derivatives of fluorescein: 1) calcein AM (<http://www.invitrogen.com/content.cfm?pageid=8012&sku=C1430>C1430, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=C3099>C3099, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=C3100MP>C3100MP), 2) BCECF AM (<http://www.invitrogen.com/content.cfm?pageid=8012&sku=B1150>B1150), 3) fluorescein diacetate (FDA, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=F1303>F1303), 4) carboxyfluorescein diacetate (CFDA, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=C1354>C1354) and 5) CellTracker Green CMFDA (5-chloromethylfluorescein diacetate, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=C2925>C2925, <http://www.invitrogen.com/content.cfm?pageid=8012&sku=C7025>C7025). Cells were incubated in 4 µM staining solutions in Dulbecco's modified eagle medium containing 10% fetal bovine serum (DMEM+) at 37°C. After incubation for 30 minutes, cell samples were immediately analyzed by flow cytometry to determine the average fluorescence per cell at time zero (0 hours). Retained cell samples were subsequently washed twice by centrifugation, resuspended in DMEM+, maintained at 37°C for 2 hours and then analyzed by flow cytometry. The decrease in the average fluorescence intensity per cell in these samples relative to the time zero samples indicates the extent of intracellular dye leakage during the 2-hour incubation period. Thus, I would recommend to analyse the samples as soon as possible or to switch to another substrate. If you would like to continue in using FDG, there are cheaper sources. Hope this is a bit of help. Best regards, Arne a.schulz@mobitec.de 2/Dear Simona, Did you play with conditions for osmotic shock, perhaps substrate doesn't get in? Also, can you proof bioactivity with X-gal staining? Beware however, passive transfer of enzyme from cell to cell can occur during osmotic shock (in my hands it always did, so I turned to GFP from then on). Veel geluk, Bruno Verhasselt Prof. dr. B. Verhasselt Ghent University Hospital Dpt. of Clinical Chemistry, Microbiology and Immunology 2P8 UZ Gent De Pintelaan 185 B-9000 Gent Tel +32-92402226 Fax +32-92404985 <mailto:Bruno.Verhasselt@UGent.be>Bruno.Verhasselt@UGent.be 3/ Hi Simona, we also want to do that experiment with spleenocytes of Rosa mice in the near future. If you get any useful comments, please share them with us. Some years ago, I did some FDG staining (MP) with thymocytes of lacZ knock in mice. The results were reproducible. As far as I can remember one of the critical steps was the loading of the dye at the appropriate temperature. So we prepared buffers at the right temperature for quick and easy transfer of the cells. But I was in the lucky situation to have a lacZ positive cell line as positive control, which is no longer available to me. Thanks in advance Steffen <mailto:stschmit@uni-mainz.de>stschmit@uni-mainz.de 4/ Hi maybe you might try indirect intracellular staining: stain your cells of interest with anti-LacZ(1st Ab) then use 2nd antibody conjugated FITC or PE but also against the first Ab. By this way you can also run FLOW to see if any LacZ positive cells are there. Is it possible to get the LacZ excited by laser? If not it might explain why FACS fails. Li <mailto:chen-22@medctr.osu.edu>chen-22@medctr.osu.edu 5/ Perhaps the substrate in not getting into your cells. M Probes sells more than one kit to help evaluate substrate loading (Detectagene vs Imagene). One uses hypotonic and one uses lipophilic loading. We've also had some luck with fixing the cells and staining with anti-LacZ antibodies. ---Dennis le FDG (Sigma F-2756, dilue en DMSO) à 200 µM, le R-(+)-Verapamil (sigma V-106 dilue en H2O) à 200 µM, et la Chloroquine diphosphate (Sigma C-6628 dilue en H2O) à 200 µM <mailto:djyoung@ucsd.edu>djyoung@ucsd.edu 6/ Marcos le FDG (Sigma F-2756, dilue en DMSO) 200 µM, R-(+)-Verapamil (sigma V-106 dilue en H2O) 200 µM, Chloroquine diphosphate (Sigma C-6628 dilue en H2O) à 200 µM Osmotic shock at 37 degrees Simona ZOMPI MD,PhD Hubrecht Laboratory Uppsalalaan, 8 3584 CT Utrecht The Netherlands Tel: +31 (0)30 212 1846 Fax: +31 (0)30 212 1801 Support Care International http://www.care.org
