>>Date: Mon, 22 Jan 2001 19:43:52 -0500 >>From: "Henry H. Wortis" <henry.wortis@tufts.edu> >>Subject: CFSE >>To: Cytometry Mailing List <cytometry@flowcyt.cyto.purdue.edu> >>Organization: Tufts University School of Medicine >>X-Accept-Language: en >>X-PMFLAGS: 34078848 0 1 31390.cnm >> >> >>Would someone with experience using CFSE, particularly with murine >>B cells, confirm >>the correct dose? The one published >>study with B cells that I found states that a dose of 10 nM/ml was >>used. This seems >>to be about 1000 fold less than the >>usual dose. >> Dear Henry, Murine B cells are excellent subjects for CFSE division tracking. I have included a list of publications using CFSE to examine B cell isotype switching, differentiation and proliferation below including some reviews, and methods for intracellular staining of cells and nuclei. Most of these papers cover the use of CFSE in in vitro applications. However, I am aware that others including the labs of Bruce Lyons, Barbara Fazekas de St Groth and Tony Basten have had very good results with transfer of labelled B cells for tracking in vivo. The concentration we use for murine B cells is 10 microMolar. I think Jhag Hasbold and I must take blame for your question. One of our papers, EJI 28:1040 has a mistake as you point out that we missed in proof - nM instead of microMolar. Papers using the CFSE technique to monitor B cell differentiation and isotype switching: 1. P.D. Hodgkin, J-H. Lee and A.B. Lyons. (1996) B cell differentiation and isotype switching is related to division cycle number. J. Exp. Med., 184: 277-281. 2. J. Hasbold, A.B. Lyons, M.R. Kehry, and P.D. Hodgkin. (1998) Cell division number regulates IgG1 and IgE switching of B cells following stimulation by CD40 ligand and IL-4. Eur. J. Immunol., 28: 1040-1051. (this one has the error in CFSE concentration) 3. M.N. McCall and P.D. Hodgkin. (1999) Switch recombination and germ-line transcription are division-regulated events in B lymphocytes. Biochim. Biophys. Acta. 1447:43-50. 4. J. Hasbold, J.S.-Y. Hong, M.R. Kehry and P.D. Hodgkin. (1999) Integrating signals from IFN-g and IL-4 by B cells: Positive and negative effects on CD40 ligand-induced proliferation, survival, and division-linked isotype switching to IgG1, IgE, and IgG2a. J. Immunol., 163:4175-4181. 5. M. Janas, P.D. Hodgkin, M. Hibbs and D. Tarlinton. (1999) Genetic evidence for Lyn as a regulator of IL-4 signalling. J. Immunol. 163:4192-4198. 6. E.K. Deenick, J. Hasbold and P.D. Hodgkin. (1999) Switching to IgG3, IgG2b and IgA is division-linked and independent revealing a stochastic framework for describing differentiation. J. Immunol., 163:4707-4714. 7. J. Hasbold, and P.D. Hodgkin (2000) Flow cytometric division tracking using nuclei. Cytometry 40:230-237. You may also be interested in a review from Bruce Lyons, Jhag Hasbold and myself that covers many of the issues associated with the use of CFSE: A.B. Lyons, J. Hasbold and P.D. Hodgkin. (2000) Flow cytometric analysis of cell division history using dilution of CFSE, a stably integrated fluorescent probe. Methods in Cell Biology - Cytometry. 63:375-398. And some papers on T cells from my lab in which you may find techniques applicable to B cells in relation to monitoring cells using intracellular staining or for making quantitative measurements of division rates. A.V. Gett and P.D. Hodgkin. (1998) Cell division regulates the T cell cytokine repertoire revealing a mechanism underlying immune class regulation. Proc. Natl. Acad. Sci. USA, 95: 9488-9493 A.V. Gett, and P.D. Hodgkin (2000) A cellular calculus for signal integration by T cells. Nature Immunology. 3:239-244. In addition here is the reference to the original paper of Lyons and Parish that introduced us all to the fascinating potential of this dye for labelling B and T cells. A.B. Lyons and CR Parish. (1994) Determination of lymphocyte division by flow cytometry. J. Immunol. Meth. 171:131-137 Best regards, Phil Hodgkin Centenary Institute of Cancer Medicine and Cell Biology Sydney Phone: (02) 9565 6121 FAX: (02) 9565 6103 email: P.Hodgkin@centenary.usyd.edu.au
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