Dear Gerhard and Tony, I agree with Gerhard that cell tracking procedures are the ideal choice for studying proliferation. In my field (exp. hematology) there are already some excellent publications in case you want to have detailed information. Just a few that I have at hand right now: What I guess is the starting article: Determination of lymphocyte division by flow cytometry. Bruce Lyons and Christopher Parish. J Immunol Methods 171, 131-137, 1994. Proliferation induced decline of primitive hematopoietic progenitor cell activity is coupled with an increase in apoptosis of ex vivo expanded CD34+ cells. Traycoff et al. Exp Hematol 26: 53-62, 1998. Following the fate of individual T cells throughout activation and clonal expansion. Andrew Wells et al. J Clin Invest 100: 3173-3183, 1997. High resolution cell division tracking demonstrates the flt3 ligand dependence of human marrow cd34+cd38- cell production in vitro. Robert Nordon et al. Br J Haematol 98: 528-539, 1997. In our hands the CFDA succinimidyl ester gives much better resolution than other products (PKH dyes) that are sold as cell trackers. Another alternative, but for this you need an UV excitation (high power Ar ion lasers or HeCd laser) is the use of the BrdUrd-Hoechst quenching technique. With this you can follow the proliferative history of cells for up to 3 to 4 cell divisions. We have published on this as well as Peter Rabinovitch's and Manfred Kubbies' group and Mike Ormerod. Details may be found in: Cell cycle analysis via BRDU-Hoechst flow cytometry- Principles and applications. Kubbies et al. in FCM:Advanced research and clinical applications, vol II, ed. Yen, CRC Press, 1989. M. Kubbies, B. Goller & D. Van Bockstaele Improved -Hoechst bivariate cell kinetic analysis by Helium-Cadmium single laser excitation. Cytometry: 13: 782 (1992) D.R. Van Bockstaele, F. Lardon, H.W. Snoeck, M.E. Peetermans, M. Kubbies BrdU-Hoechst-Ethidium bromide (EB) quenching technique for studying kinetics of hematopoiesis. Blood 80: 289-291 (1992) M. Kubbies, B. Goller & D. Van Bockstaele Improved -Hoechst bivariate cell kinetic analysis by Helium-Cadmium single laser excitation. Cytometry: 13: 782 (1992) - paper F. Lardon, D.R. Van Bockstaele, H.W. Snoeck, M.E. Peetermans Quantitative cell cycle progression analysis of the first 3 successive cell cycles of G-CSF and/or GM-CSF stimulated human CD34+ bone marrow cells in relation to their colony formation. Blood 88: 3211-3216 (1993) Filip Lardon, Hans-W. Snoeck, Griet Nijs, Marc Lenjou, Marc E. Peetermans, Inez Rodrigus, Zwi N. Berneman, Dirk R. Van Bockstaele. Transforming growth factor-b regulates the cell cycle status of interleukin-3 plus interleukin-1, stem cell factor or interleukin-6 stimulated CD34+ human hematopoietic progenitor cells through different cell kinetic mechanisms depending on the applied stimulus. Exp. Hematol. 22: 903-909 (1994) F Lardon, H-W Snoeck, L Haenen, M Lenjou, G Nijs, SFA Weekx, PCF Van Ranst, ZN Berneman and DR Van Bockstaele. The combined effects of all-trans retinoic acid and TGF-b on the initial proliferation of normal human bone marrow progenitor cells. Leukemia 10: 1937-1943, 1996 I hope this can be of help. Best Regards, Dirk Prof. Dirk Van Bockstaele, PhD Head Flow Cytometry Facility Laboratory of Hematology Antwerp University Hospital Belgium > ---------- > Van: Gerhard Nebe-von-Caron[SMTP:Gerhard.Nebe-von-Caron@Unilever.com] > Verzonden: maandag 16 augustus 1999 12:02 > Aan: Cytometry Mailing List > Onderwerp: RE: Opinions on proliferation by flow > > <<Bestand: ATT31156.ATT>> > The best way to track the situation is to covalently label the cells and > then > do your antibody staining. There was an excellent paper given about the > theorie > of clonal expansion on the Sam Latt conference (you all missed out on an > excellent conference if you were not there) by Phillip Hodgkin from > Sydney. He > combined CFDA-succinimidylester labelling with antibody staining and > demonstrated the time dependancy of clonal expansion in a mathematical > model. > Pitty there isn't a printed summary, but I look forward to the paper. > > > Gerhard > > -----Original Message----- > From: Tony Schountz [SMTP:tschount@mesastate.edu] > Sent: Friday, August 13, 1999 5:53 PM > To: Cytometry Mailing List > Subject: Opinions on proliferation by flow > > > I'd like opinions on the use of flow for the detection of T cell > proliferation. I know that bromodeoxyuridine is an established method, > but right now out of my reach ($$). My thought is to use anti-CD3, -CD4 > and/or -CD8 on bulk lymph node cultures stimulated with or without > antigen to get changes in total numbers of cells. > > Thanks, > > Tony > > -- > Tony Schountz, Ph.D. > Department of Biological Sciences > Mesa State College > mailto:tschount@mesastate.edu > > >
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