I agree that for cell sizing the Coulter really is the only option. Nucleated cell probably give more interference in laser light scattering techniques than possible with a Coulter. To use a flow cytometer to get an idea of cell volume it would probably be better to use a protein stain eg: FITC rather than FSC. FSC does not correlate very well with Coulter volume especially when the cells are under- going changes (resulting in differing scattering properties). Protein content however does generally correlate directly with cell size during balanced growth. (Frame and Hu, 1990, Biotechnol. Bioeng.36: 191-197) Measuring the Coulter volume one should always be sure to be careful of the Osmometer effect. Especially when the sample is derived from a system of unknown or undefined osmolality. eg:diluting cells in a solution of higher osmolality than the source will generally lead to immediate shrinkage and measurement of a volume which doesn't truly represent that of the source. (Rosinski, 2000, Biotechnol. Prog. 16: 782-785) Incidentally, regarding Jim's message I think smaller plastic beads actually scatter light more widely than larger ones according to Mie Theory. What is measured in the FSC PMT is probably a stronger signal for larger particles because they FAIL to scatter the incident light as much as smaller particles. Perhaps someone can clarify? Matt Rosinski _______________________________________________________ Matthew Rosinski The University of Queensland Department of Chemical Engineering College Rd St Lucia Q 4072 Australia Ph: (07) 3365 8392/4352 _____________________________________________________ -----Original Message----- From: James Weaver 301-594-5879 FAX 301-594-3037 [SMTP:WEAVER@CDER.FDA.GOV] Sent: Friday, October 13, 2000 3:55 AM To: Cytometry Mailing List Subject: Re: Cell Volume Revisited Despite what is written in various places, flow cytometry can only be used for size measurement on homogenous particles such as plastic beads. It does not work for particles with complex optics such as cells. With beads, increasing size leads to increased light scatter. With cells the situation is quite different, a treatment that resulted in a 30% change in cell volume caused a light scatter decrease in a lymphoid cell line and no change in light scatter from NIH3T3 cells. Coulter volume measurements are regarded as the gold standard of cell volume measurements. I have done cell volume measurements on nucleated cells in the past with no problems. If you are not seeing changes in cell volume, than either the treatment is not causing a change or the instrument needs repair. -Jim Weaver ************************************************* * * * James L. Weaver Ph.D. * * Division of Applied Pharmacology Research * * Office of Testing & Research * * CDER MOD-1, FDA * * 8301 Muirkirk Rd, Laurel MD 20708 * * * * Phone: 301-594-5879 * * Fax: 301-594-3037 * * Email:WEAVER@CDER.FDA.GOV * * * *************************************************
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