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Rapid Clonal Growth Measurements at the Single-Cell Level: Gel Microdroplets and Flow Cytometry

Abstract

We describe a new, general method for rapidly measuring clonal growth of large numbers of individual members of a cell population. This method is based on microculture of individual colony-forming units in gel microdrops (GMDs; here aga-rose; 20 to 90μ in diameter), which are sufficiently robust to be handled much like cells, and diffusionally transparent for molecules of interest. Flow cytometry provides rapid measurements of GMD-entrapped microcolonies, and permits sub-population analysis. Here the method is demonstrated with mammalian, fungal and bacterial species. Additional results illustrate rapid determination of a drug-resistant subpopulation in a mixed species sample, and nutrient sensitivity for a murine hybridoma.

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References

  1. 1

    Lennette, E.H. (Ed.). 1985. Manual of Clinical Microbiology, Fourth Edition. American Society for Microbiology, Washington D.C.

  2. 2

    Freshney, R.I. 1987. Culture of Animal Cells: A Manual of Basic Technique, Second Edition. Alan R. Liss, New York.

  3. 3

    Hattori, T. 1988. Quantitative and Environmental Aspects. Brock/Springer, Madison, WI.

  4. 4

    Weaver, J.C., Seissler, P.E., Threefoot, S.A., Lorenz, J.W., Huie, T., Rodrigues, R. and Klibanov, A.M. 1984. Microbiological measurements by immobilization of cells within small volume elements. Ann. N.Y. Acad Sci. 434: 363–372.

  5. 5

    Weaver, J.C. 1986. Gel microdroplets for microbial measurement and screening: Basic principles. Biotech. and Bioengr. Symp. 17: 185–195.

  6. 6

    Williams, G.B., Threefoot, S.A., Lorenz, J.W., Bliss, J.G., Weaver, J.C., Demain, A.L. and Klibanov, A.M. 1987. Rapid detection of E. coli immobilized in gel microdroplets. Ann N.Y. Acad. Sci. 501: 350–353.

  7. 7

    Weaver, J.C., Williams, G.B., Klibanov, A.M. and Demain, A.L. 1988. Gel microdroplets: Rapid detection and enumeration of individual microorganisms by their metabolic activity. Bio/Technology 6: 1084–1089.

  8. 8

    Williams, G.B., Weaver, J.C. and Demain, A.L. 1990. Rapid microbial detection and enumeration using gel microdroplets: Colorimetric versus fluorescent indicator systems. J. Clin. Microbiol. 28: 1002–1008.

  9. 9

    Nir, R., Lamed, R., Gueta, L. and Sahar, E. 1990. Single-cell entrapment and microcolony development within uniform microspheres amenable to flow cytometry. Appl. Environ. Microbiol. 56: 2870–2875.

  10. 10

    Powell, K.T. and Weaver, J.C. 1990. Gell microdroplets and flow cytometry: Rapid determination of antibody secretion by individual cells within a cell population. Bio/Technology 8: 333–337.

  11. 11

    Scott, C.D. 1987. Immobilized cells: A review of recent literature. Enz. Microb. Technol. 9: 66–73.

  12. 12

    Nilsson, K., Scheirer, W., Katlinger, H.W.D. and Mosbach, K. 1987. Entrapment of animal cells. Methods in Enzymol. 135: 399–410.

  13. 13

    Akporiaye, E.T., Kudalore, M., Kraemer, P.M. and Stewart, C.C. 1988. Isolation and reactivity of host effectors associated with the manifestation of concomitant tumor immunity. Cancer Res. 48: 1153–1158.

  14. 14

    Grunewald, W. 1973. Accuracy and errors of the PO2 measurement by means of the platinum electrode and its calibration in vivo, p. 309–320. In: Modern Techniques in Physiological Sciences, J. F. Gross, R. Kaufmann and E. Wetterer (eds.) Academic Press, New York.

  15. 15

    Bliss, J.G. 1990. Rapid determination of antimicrobial susceptibility using gel microdroplets and flow cytometry, Ph.D. Thesis, M.I.T.

  16. 16

    Powell, K.T. 1989. Mammalian cell clonal growth and secretion measurements using gel microdroplets and flow cytometry, Ph.D. Thesis, M.I.T.

  17. 17

    Lundak, R.L. and Raidt, D.J. 1973. Cellular immune response against tumor cells. Cellular Immunol. 9: 60–66.

  18. 18

    Parham, P. and Bodmer, W.F. 1978. Monoclonal antibody to a human histocompatibility alloantigen, HLA-A2. Nature 276: 397–399.

  19. 19

    Shapiro, H.M. 1988. Practical Flow Cytometry, Second Edition. A. R. Liss, New York.

  20. 20

    Melamed, E.R., Lindmo, T. and Mendelsohn, M.L. 1990. Flow Cytometry and Sorting, Second Edition. Wiley-Liss, New York.

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