Summary
We have prepared fluorescein isothiocyanate (FITC) conjugates of cationised ferritin (CF) and have investigated the usefulness of this CF-FITC to measure the negative cell surface charge of mouse bone marrow cells by flow cytometry. CF-FITC conjugates of low fluorochrome to protein ratios (F/P ratio) gave insufficient fluorescence and/or formed large aggregates when stored. CF-FITC conjugates of high F/P ratios (above 25) bound specifically to bone marrow cells, giving sufficient fluorescence, the intensity of which differed for the different cell types. When stored at −20° C the CF-FITC was stable and could be used over prolonged periods. CF-FITC could be used to selectively enrich for pluripotent stem cells (CFU-S) and granulocyte/macrophage progenitors (CFU-C) by fluorescence activated cell sorting (FACS), although the CF-FITC binding to CFU-S and CFU-C was unexpectedly low. No correlation between CF-FITC fluorescence, cell size and electrophoretic mobility (EMP) was observed of bone marrow cells fractionated by free flow electrophoresis. Neuraminidase treatment to remove negatively charged sialic acid groups from the cell surface resulted in an increased binding of CF-FITC, although the EPM was decreased. The biotin conjugate of CF bound to bone marrow cells and could be visualised by avidin-FITC. The relative fluorescence intensity for the individual cell types showed a good correlation with the cell surface charge as determined by the EPM of the different cell types.
The mechanism of binding CF-FITC to the cell surface was not by electrostatic interaction of the negative cell surface and positively charged CF because CF-FITC of F/P ratios of above 20 was negatively charged. This has been shown by theoretical calculations and determination of the pI of CF-FITC by iso-electric focussing. Binding of CF-FITC to the cell surfaces was probably caused by hydrophobic interaction between bound fluorescein molecules and lipid domains in the cell surface membrane aided by some ionic interaction. CF-biotin is still positively charged and is probably bound through electrostatic interactions with negatively charged cell surface groups. The indirect detection of bound CF-biotin with avidin-FITC of high F/P ratio results in a high fluorescence signal, which is a measure of the negative cell surface charge density, in the FACS.
Similar content being viewed by others
References
Baines P, Visser JWM (1983) Analysis and separatin of murine bone marrow stem cells by H33342 fluorescence-activated cell sorting. Exp Hematol 11:701–708
Bauman JGJ, Mulder AH, Engh GJ van den (1985) The effect of surface antigen labeling on spleen colony formation: comparison of the indirect immunofluorescence and the biotin-avidin methods. Exp Hematol 13:760–767
Bauman JGJ, Wagemaker G, Visser JWM (1986) A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and commited progenitors. J Cell Physiol (in press)
Bol SJL (1980) The recognition of early developmental stages in haemopoiesis. Thesis, Eramus University of Rotterdam, Rotterdam, The Netherlands
Bol S, Vliet M van (1980) Characterization and enrichment of early hemopoietic cells by use of free-flow electrophoresis. In: Baum SJ, Ledney GD, Bekkum DW van (eds) Experimental hematology today 1980. S Karger, Basel, pp 327–339
Bol S, Vliet M van, Slingerland V van (1981) Electrophoretic mobility properties of murine hemopoietic cells in different stages of development. Exp Hematol 9:431–443
Butman BT, Bourguignon GJ, Bourguignon LYW (1980) Lymphocyte capping induced by polycationized ferritin. J Cell Physiol 105:7–15
Dalen JPR van, Haaijman JJ (1974) Determination of the molar absorbance coefficient of bound tetramethyl rhodamine isothiocyanate relative to fluorescein isothiocyanate. J Immunol Methods 5:103–106
Danon D, Goldstein Y, Marikovsky Y, Skutelsky E (1972) Use of cationized ferritin as a label of negative charges on cell surfaces. J Ultrastruct Res 38:500–510
Engh G van den, Visser J, Bol S, Trask B (1980) Concentration of hemopoietic stem cells using a light-activated cell sorter. Blood Cells 6:609–623
Engh G van den, Bauman J, Mulder D, Visser J (1983) Measurement of antigen expression of hemopoietic stem cells and progenitor cells by fluorescence activated cell sorting. In: Killman SvAA, Cronkite EP, Muller-Berat CN (eds) Haemopoietic stem cells. Munksgaard, Copenhagen, pp 59–74
Hannig K, Wirth H, Meyer BH, Zeiller K (1975) Free flow electrophoresis. I. Theoretical and experimental investigations on the influence of mechanical and electrokinetic variables on the efficiency of the method. Hoppe-Seyler's Z Physiol Chem 356:1209
Horan PK, Wheeless LL Jr (1977) Quantitative single cell analysis and sorting. Science 198:149–157
King CA, Preston TM (1977) Fluoresceinated cationized ferritin as a membrane probe for anionic sites at the cell surface. FEBS Lett 73:59–63
Pimenta PFP, Souza W de (1982) Surface charge of cosinophils. Binding of cationic particles and measurement of cellular electrophoretic mobility. Histochemistry 74:569–576
Skutelsky E, Hardy B (1976) Regeneration of plasmalemma and surface properties in macrophages. Exp Cell Res 101:337–345
Steinbach G, Mayersbach H von (1976) Characterisation of fluorescein isothiocyanate. II. Absorption and fluorescence after conjugation to human- and rabbit-gamma-globulin and bovine serum albumin. Acta Histochem 55:110–123
Steinkamp JA (1984) Flow cytometry. Rev Sci Instrum 55:1375–1400
Thürauf N, Dermietzel R (1982) An improved technique for preparing polycationic ferritin derivatives. Stain Techn 57:171–175
Till JE, McCullough EA (1961) A direct measurement of the radiation sensitivity of normal mouse bone marrow cells. Radiat Res 14:213–222
Trask BJ, Engh GJ van den (1980) Antigen expression of CFU-S determined by light activated cell sorting. In: Baum SJ, Ledney GD, Bekkum DW van (eds) Experimental hematology today 1980. S Karger, Basel, pp 299–307
Valet G, Bamberger S, Hofman H, Schindler R, Ruhenstroth-Bauer G (1979) Flow cytometry as a new method for the measurement of electrophoretic mobility of erythrocytes using membrane charge staining by fluoresceinated polycations. J Histochem Cytochem 27:342
VanDilla MA, Dean PN, Laerum OD, Melamed MR (eds) (1985) Flow cytometry: Instrumentation and data analysis. Academic Press, London
Visser JWM, Bol SJL (1981) A two-step procedure for obtaining 80-fold enriched suspensions of murine pluripotent hemopoietic stem cells. Stem Cells 1:240–249
Visser JWM, Jongeling AAM, Tanke HJ (1979) Intracellular pH determination by fluorescence measurements. J Histochem Cytochem 27:32–35
Visser JWM, Engh GJ van den, Bekkum DW van (1980) Light scattering properties of murine hemopoietic cells. Blood Cells 6:391–407
Visser JWM, Bauman JGJ, Mulder AH, Eliason JF, Leeuw AM de (1984) Isolation of murine pluripotent hemopoietic stem cells. J Exp Med 59:1576–1590
Author information
Authors and Affiliations
Additional information
In honour of Prof. P. van Duijn
Rights and permissions
About this article
Cite this article
Bauman, J.G.J., Bouwman, E. The use of cationized ferritin to measure cell surface charge of mouse bone marrow cells by flow cytometry. Histochemistry 84, 454–461 (1986). https://doi.org/10.1007/BF00482978
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00482978