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Murphy Lab - Flow Cytometry


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Murphy Lab Protocols


Frequently Asked Questions

  • Has anyone used flow cytometry or sorting with individual vesicular organelles?

Answers

  • Has anyone used flow cytometry or sorting with individual vesicular organelles ? Yes... an annotated bibliography is included below.

1. R. F. Murphy (1985) Analysis and isolation of endocytic vesicles by flow cytometry and sorting: Demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis. Proc. Natl. Acad. Sci. U.S.A. 82:8523-8526.

  • Detection of endocytic vesicle subpopulations using a fluorescent fluid-phase marker (Figs. 1 and 2)
  • Detection of endocytic vesicles using a fluorogenic substrate (Fig. 3)
  • Sorting of endocytic vesicles (Fig. 4)

2. R.F. Murphy and M. Roederer (1986) Flow cytometric analysis of endocytic pathways. In: Applications of Fluorescence in the Biomedical Sciences, D.L. Taylor, A.S. Waggoner, R.F. Murphy, F. Lanni, R. Birge (eds.), Alan R. Liss, Inc., New York, pp. 545-566.

  • Normalization of vesicle fluorescence measurements using side scatter (Fig. 7)

3. R.F. Murphy (1988) Processing of endocytosed material. Adv. Cell Biol. 2:159-180.

  • Two-color analysis of endocytic vesicles using two fluid-phase markers (Fig. 4)
  • Correlation of flow cytometric analysis with density gradient fraction (and incorporation of fraction number as a "flow cytometric parameter" to allow display of contour maps, etc.) (also in Fig. 4)

4. R.B. Wilson and R.F. Murphy (1989) Flow-cytometric analysis of endocytic compartments. Methods Cell Biol. 31:293-317.

  • Description of bead calibration for SOFA (Fig. 6)
  • Detection of in vitro acidification of endocytic vesicles using acridine orange (Fig. 7)

5. R.F. Murphy, M. Roederer, D.M. Sipe, C.C. Cain, and R. Bowser (1989) Determination of the biochemical characteristics of cndocytic compartments by flow cytometric and fluorometric analysis of cells and organelles. In: Flow Cytometry: Advanced Research and Clinical Applications, Volume II, A. Yen (ed.), CRC Press, Boca Raton, FL, pp. 221-254.

  • Detection of organelles with Nile Red (combined with density gradient fractionation) (Fig. 11)
  • Detection of endocytic vesicle subpopulations using a receptor-mediated probe (Fig. 13)

6. M.L. Zeidel, T. Hammond, B. Botelho, and H. W. Harris, Jr. (1992) Functional and structural characterization of endosomes from toad bladder epithelial cells. Am. J. Physiol. 263:F62-F76.

7. F.G. van der Goot, A. Seigneur, J.C. Gaucher, and P. Ripoche (1992) Flow cytometry and sorting of amphibian bladder endocytic vesicles containing ADH-sensitive water channels. J. Membr. Biol. 128:1330139.

8. R.M. Guasch, C. Guerri, J.E. O'Connor (1993) Flow cytometric analysis of concanavalin A binding to isolate Golgi fractions from rat liver. Exp. Cell Res. 207:136-141.

9. T.G. Hammond, R.R. Majewski, D.J. Morre, K. Schell, L.W. Morrissey (1993) Forward scatter pulse width signals resolve multiple populations of endosomes. Cytometry 14:411-420.

10. T.G. Hammond, D.J. Morre, H.W. Harris, Jr., and M.L. Zeidel (1993) Isolation of highly purified, functional endosomes from toad urinary bladder. Biochem. J. 295:471-476.

11. H.W. Harris, Jr., M.L. Zeidel, I. Jo, and T.G. Hammond (1994) Characterization of purified endosomes containing the antidiuretic hormone-sensitive water channel from rat renal papilla. J. Biol. Chem. 269:11993-12000.




Last Updated: 01 Dec 2004




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