From: Tom Frey (Tom_Frey@bdis.com)
Date: Tue Feb 24 1998 - 15:13:09 EST
Jodi While Brian makes a useful suggestion for cells, the protocol that you are using will probably prevent it from working. The NP-40 you use to make nuclei will probably also extract any membrane label. If you feel that you MUST make nuclei you will likely be limited to shooting (in the relative dark) for an antibody that recognizes an internal antigen retained by the process. A species specific nuclear structural protein might work, or if you are lucky some type of intermediate filament in the stromal cells might be preserved. Usually this type of prep leaves enough cytokeratin in epithelial cells, but my recollection is that stromal cells are endothelial or mesothelial - perhaps a vimentin or something. A less random thing to do might be to use live cells, UV exictation, and Hoechst 33342. You could try running the cells and see if they are distinguishable by scatter when unfixed to determine if this is a useful method to pursue. (The suggestion of membrane or MitoTrackers would also work in this system.) If you don't have UV - or if you think that H342 doesn't give suitable CVs - you could try ethanol fixation. Some surface eptiopes are preserved by this process, you might be able to use CD38 or some mouse stromal marker, or even cytoplamic Ig. Just some thoughts. Tom Frey _______________________________________________________________________________ Subject: RE: Cell cycle analysis on myeloma cells From: "Newsom; Brian S." <bsnewsom@msmail.his.tch.tmc.edu> at INTERNET Date: 2/20/98 2:47 PM Jodi, One thing you might try is to label the myeloma or fibroblast cells with a lipid dye such as PKH. These dyes remain intact in the cells for up to 100 days depending on cycle rate. As the cell divides and becomes two daughter cells the dye intensity is cut in half due to division of the membrane between the two new cells. I would say choose the cell that cycles the slowest and stain it with this dye. It comes in green and red and maybe other colors and works well and does not effect cell viability. It should make it easy to distinguish your two cell types. Brian Newsom Flow Cytometry Specialist Baylor College of Medicine Shell Center for Gene Therapy You Wrote: Dear Colleagues, I am submitting this question for a researcher who uses my core facility. Objective: To study the cell cycle in human myeloma cell line(s) plated on top of a murine stroma cell line. Hypothesis: Upon adherence of myeloma cells to stromal cells, the cell cycle distribution of myeloma cells may be affected. Procedure: Human myeloma cells are plated onto the monolayer of gamma-irradiated murine stromal fibroblasts. After 2 hours, non-adherent myeloma cells are removed. Adherent cells are further cultured for up to 72 hours. After completion of co-culture, cells are lysed/stained in situ with a buffer containing 0.1% Na citrate, 0.3% NP-40, 100ug/ml RNase A, and 50ug/ml propidium iodide, vortexed at a high speed for 1 min., and kept on ice until flow analysis. Problems: We cannot consistantly discriminate myelomas from stromas based on their morphologies. In each of four experiments, the stromas have varied in size and complexity (why I don't know, and the researcher cannot explain this issue). However, the myelomas have consistantly shown the same size and complexity. Therefore, we cannot use morphology to distinquish myeloma from stroma because I cannot single out the myeloma with a gate. The researcher attemped to pre-stain the stroma cells with a SYTO dye, but the fluorescence of this dye was completely quenched upon PI staining as above. (Has anyone seen this happen or have an explanation for it?) The initial reasoning for doing this was to see if we could gate out the green fluorescent stromal cells from the PI stained myeloma cells. (I wasn't sure if this was going to work, but I thought it was worth a try.) Questions: Has anyone successfully done anything like this in the past who might be so gracious as to share his/her protocol? If not, does anyone have any suggestions as to what my fraid researcher might try? Sorting the two populations does not seem like a "viable" option because the myeloma are already in a fragile state after culture. Any response is greatly appreciated at this point. Thanks in advance, Jodi L. Kroeger (kroegerjl@moffitt.usf.edu) Moffitt Cancer Center and Research Institute Tampa, Florida Flow Cytometry Core Facility
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