Quantification of Phagocytosis in Human Neutrophils by Flow Cytometry

Michael Heinzelmann; Sarah Appel Gardner; Mark Mercer-Jones; Amy J. Roll; Hiram C. Polk Jr.

Michael Heinzelmann, Sarah Appel Gardner, Mark Mercer-Jones, Amy J. Roll, Hiram C. Polk Jr.

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Keywords: Phagocytosis, Flow cytometry, Quantitative analysis, Kinetics

JOURNAL FREE ACCESS

1999 Volume 43 Issue 6 Pages 505-512

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Published: June 20, 1999 Received: November 30, 1998 Available on J-STAGE: March 17, 2008 Accepted: March 01, 1999 Advance online publication: - Revised: -
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Date of correction: March 17, 2008 Reason for correction: - Correction: ABSTRACT Details: Wrong : Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeted extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1:1, 1:10, 1:100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.
Right : Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1 : 1, 1 : 10, 1 : 100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

Date of correction: March 17, 2008 Reason for correction: - Correction: DTPUB Details: Right : 19990620

Date of correction: March 17, 2008 Reason for correction: - Correction: AUTHOR Details: Wrong : Michael Heinzelmann¹⁾, Sarah Appel Gardner¹⁾, Mark Mercer-Jones¹⁾, Amy J. Roll¹⁾, Hiram C. Polk Jr.¹⁾
Right : Michael Heinzelmann¹⁾, Sarah Appel Gardner¹⁾, Mark Mercer-Jones¹⁾, Amy J. Roll¹⁾, Hiram C. Polk Jr.²⁾

Date of correction: March 17, 2008 Reason for correction: - Correction: AFFILIATION Details: Wrong :
1) The Price Institute of Surgical Research, Department of Surgery, University of Louisville School of Medicine

Right :
1) The Price Institute of Surgical Research, Department of Surgery, University of Louisville School of Medicine
2) Department of Surgery, University of Louisville, Louisville

Date of correction: March 17, 2008 Reason for correction: - Correction: CITATION Details: Wrong : 1) Bjerknes, R., and Bassoe, C.F. 1983. Human leukocyte phagocytosis of zymosan particles measured by flow cytometry. Acta Pathol. Microbiol. Immunol. Scand. 91: 341-348.
2) Bjerknes, R., Bassoe, C.F., Sjursen, H., Laerum, O.D., and Solberg, C.O. 1989. Flow cytometry for the study of phagocyte functions. Rev. Infect. Dis. 11: 16-33.
3) Boyum, A. 1968. A one-stage procedure for isolation of granulocytes and lymphocytes from human blood: general sedimentation properties of white blood cells in a 1g gravity field. Scand. J. Clin. Lab. Invest. (Suppl.) 97: 51-76.
4) Campbell, P.A., Canono, B.P., and Drevets, D.A. 1994. Measurement of bacterial ingestion and killing by macrophages, p. 12-13. In Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Storber, W. (eds), Current protocols in immunology, John Wiley & Sons, Inc., New York.
5) Cantinieaux, B., Hariga, C., Courtoy, P., Hupin, J., and Fondu, P. 1989. Staphylococcus aureus phagocytosis: a new cytofluorometric method using FITC and paraformaldehyde. J. Immunol. Methods 121: 203-208.
6) Drevets, D.A., and Campbell, P.A. 1991. Macrophage phagocytosis: use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J. Immunol. Methods 142: 31-38.
7) Dunn, P.A., and Tyrer, H.W. 1981. Quantitation of neutrophil phagocytosis, using fluorescent latex beads: correlation of microscopy and flow cytometry. J. Lab. Clin. Med. 98: 374-381.
8) Fattorossi, A., Nisini, R., Pizzolo, J.G., and D'Amelio, R. 1989. New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis. Cytometry 10: 320-325.
9) Glasser, L., and Fiederlein, R.L. 1990. The effect of various cell separation procedures on assays of neutrophil function: a critical appraisal. Am. J. Clin. Pathol. 93: 662-669.
10) Hampton, M.B., Vissers, M.C., and Winterbourn, C.C. 1994. A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils. J. Leukoc. Biol. 55: 147-152.
11) Hed, J. 1977. The extinction of fluorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis. FEMS Lett. 1: 357-361.
12) Hed, J. 1986. Methods for distinguishing ingested from adhering particles. Methods Enzymol. 132: 198-204.
13) Hed, J., Hallden, G., Johansson, S.G., and Larsson, P. 1987. The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J. Immunol. Methods 101: 119-125.
14) Martin, E., and Bhakdi, S. 1992. Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes. J. Clin. Microbiol. 30: 2246-2255.
15) Matsuda, J., Tsukamoto, M., Saitoh, N., Gohchi, K., Jimbo, S., Shibui, T., and Moriya, K. 1993. Polymorphonuclear leucocyte function tests: a comparison of cytochrome C reduction and flow cytometric analysis. Br. J. Biomed. Sci. 50: 60-63.
16) Ogle, J.D., Noel, J.G., Sramkoski, R.M., Ogle, C.K., and Alexander, J.W. 1988. Phagocytosis of opsonized fluorescent microspheres by human neutrophils. A two-color flow cytometric method for the determination of attachment and ingestion. J. Immunol. Methods 115: 17-29.
17) Perticarari, S., Presani, G., and Banfi, E. 1994. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Methods 170: 117-124.
18) Peterson, P.K., Verhoef, J., Schmeling, D., and Quie, P.G. 1977. Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. J. Infect. Dis. 136: 502-509.
19) Steinkamp, J.A., Wilson, J.S., Saunders, G.C., and Stewart, C.C. 1982. Phagocytosis: flow cytometric quantitation with fluorescent microspheres. Science 215: 64-66.
20) Stewart, C.C., Lehnert, B.E., and Steinkamp, J.A. 1986. In vitro and in vivo measurement of phagocytosis by flow cytometry. Methods Enzymol. 132: 183-192.
21) Sveum, R.J., Chused, T.M., Frank, M.M., and Brown, E.J. 1986. A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis. J. Immunol. Methods 90: 257-264.
22) Venaille, T.J., Misso, N.L., Phillips, M.J., Robinson, B.W., and Thompson, P.J. 1994. Effects of different density gradient separation techniques on neutrophil function. Scand. J. Clin. Lab. Invest. 54: 385-391.
23) Verhoef, J., Peterson, P.K., and Quie, P.G. 1977. Kinetics of staphylococcal opsonization, attachment ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [³H] thymidine labeled bacteria. J. Immunol. Methods 14: 303-311.
24) Watson, F., Robinson, J.J., and Edwards, S.W. 1992. Neutrophil function in whole blood and after purification: changes in receptor expression, oxidase activity and responsiveness to cytokines. Biosci. Rep. 12: 123-133.
25) White-Owen, C., Alexander, J.W., Sramkoski, R.M., and Babcock, G.F. 1992. Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J. Clin. Microbiol. 30: 2071-2076.
26) Wilson, R.M., Galvin, A.M., Robins, R.A., and Reeves, W.G. 1985. A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes. J. Immunol. Methods 76: 247-253.

Right : 1) Bjerknes, R., and Bassoe, C.F. 1983. Human leukocyte phagocytosis of zymosan particles measured by flow cytometry. Acta Pathol. Microbiol. Immunol. Scand. 91 : 341-348.
2) Bjerknes, R., Bassoe, C.F., Sjursen, H., Laerum, O.D., and Solberg, C.O. 1989. Flow cytometry for the study of phagocyte functions. Rev. Infect. Dis. 11 : 16-33.
3) Boyum, A. 1968. A one-stage procedure for isolation of granulocytes and lymphocytes from human blood : general sedimentation properties of white blood cells in a 1 g gravity field. Scand. J. Clin. Lab. Invest. (Suppl.) 97 : 51-76.
4) Campbell, P.A., Canono, B.P., and Drevets, D.A. 1994. Measurement of bacterial ingestion and killing by macrophages, p. 12-13. In Coligan, J.E., Kruisbeek, A.M., Margulies, D.H., Shevach, E.M., and Storber, W. (eds), Current protocols in immunology, John Wiley & Sons, Inc., New York.
5) Cantinieaux, B., Hariga, C., Courtoy, P., Hupin, J., and Fondu, P. 1989. Staphylococcus aureus phagocytosis : a new cytofluorometric method using FITC and paraformaldehyde. J. Immunol. Methods 121 : 203-208.
6) Drevets, D.A., and Campbell, P.A. 1991. Macrophage phagocytosis : use of fluorescence microscopy to distinguish between extracellular and intracellular bacteria. J. Immunol. Methods 142 : 31-38.
7) Dunn, P.A., and Tyrer, H.W. 1981. Quantitation of neutrophil phagocytosis, using fluorescent latex beads : correlation of microscopy and flow cytometry. J. Lab. Clin. Med. 98 : 374-381.
8) Fattorossi, A., Nisini, R., Pizzolo, J.G., and D'Amelio, R. 1989. New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis. Cytometry 10 : 320-325.
9) Glasser, L., and Fiederlein, R.L. 1990. The effect of various cell separation procedures on assays of neutrophil function : a critical appraisal. Am. J. Clin. Pathol. 93 : 662-669.
10) Hampton, M.B., Vissers, M.C., and Winterbourn, C.C. 1994. A single assay for measuring the rates of phagocytosis and bacterial killing by neutrophils. J. Leukoc. Biol. 55 : 147-152.
11) Hed, J. 1977. The extinction of fluorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis. FEMS Lett. 1 : 357-361.
12) Hed, J. 1986. Methods for distinguishing ingested from adhering particles. Methods Enzymol. 132 : 198-204.
13) Hed, J., Hallden, G., Johansson, S.G., and Larsson, P. 1987. The use of fluorescence quenching in flow cytofluorometry to measure the attachment and ingestion phases in phagocytosis in peripheral blood without prior cell separation. J. Immunol. Methods 101 : 119-125.
14) Martin, E., and Bhakdi, S. 1992. Flow cytometric assay for quantifying opsonophagocytosis and killing of Staphylococcus aureus by peripheral blood leukocytes. J. Clin. Microbiol. 30 : 2246-2255.
15) Matsuda, J., Tsukamoto, M., Saitoh, N., Gohchi, K., Jimbo, S., Shibui, T., and Moriya, K. 1993. Polymorphonuclear leucocyte function tests : a comparison of cytochrome C reduction and flow cytometric analysis. Br. J. Biomed. Sci. 50 : 60-63.
16) Ogle, J.D., Noel, J.G., Sramkoski, R.M., Ogle, C.K., and Alexander, J.W. 1988. Phagocytosis of opsonized fluorescent microspheres by human neutrophils. A two-color flow cytometric method for the determination of attachment and ingestion. J. Immunol. Methods 115 : 17-29.
17) Perticarari, S., Presani, G., and Banfi, E. 1994. A new flow cytometric assay for the evaluation of phagocytosis and the oxidative burst in whole blood. J. Immunol. Methods 170 : 117-124.
18) Peterson, P.K., Verhoef, J., Schmeling, D., and Quie, P.G. 1977. Kinetics of phagocytosis and bacterial killing by human polymorphonuclear leukocytes and monocytes. J. Infect. Dis. 136 : 502-509.
19) Steinkamp, J.A., Wilson, J.S., Saunders, G.C., and Stewart, C.C. 1982. Phagocytosis : flow cytometric quantitation with fluorescent microspheres. Science 215 : 64-66.
20) Stewart, C.C., Lehnert, B.E., and Steinkamp, J.A. 1986. In vitro and in vivo measurement of phagocytosis by flow cytometry. Methods Enzymol. 132 : 183-192.
21) Sveum, R.J., Chused, T.M., Frank, M.M., and Brown, E.J. 1986. A quantitative fluorescent method for measurement of bacterial adherence and phagocytosis. J. Immunol. Methods 90 : 257-264.
22) Venaille, T.J., Misso, N.L., Phillips, M.J., Robinson, B.W., and Thompson, P.J. 1994. Effects of different density gradient separation techniques on neutrophil function. Scand. J. Clin. Lab. Invest. 54 : 385-391.
23) Verhoef, J., Peterson, P.K., and Quie, P.G. 1977. Kinetics of staphylococcal opsonization, attachment ingestion and killing by human polymorphonuclear leukocytes : a quantitative assay using [³H] thymidine labeled bacteria. J. Immunol.Methods 14 : 303-311.
24) Watson, F., Robinson, J.J., and Edwards, S.W. 1992. Neutrophil function in whole blood and after purification : changes in receptor expression, oxidase activity and responsiveness to cytokines. Biosci. Rep. 12 : 123-133.
25) White-Owen, C., Alexander, J.W., Sramkoski, R.M., and Babcock, G.F. 1992. Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J. Clin. Microbiol. 30 : 2071-2076.
26) Wilson, R.M., Galvin, A.M., Robins, R.A., and Reeves. W.G. 1985. A flow cytometric method for the measurement of phagocytosis by polymorphonuclear leucocytes. J. Immunol. Methods 76 : 247-253.

Abstract

Phagocytosis represents a central element of the host response to microbial invasion. We describe a flow cytometric method for measuring the kinetics of phagocytosis of two bacteria by human polymorphonuclear leukocytes (PMNs). Over a 60-min period, isolated human PMNs were exposed to Staphylococcus aureus (rapidly phagocytosed) and Klebsiella pneumoniae (slowly phagocytosed). This method distinguished adherent from ingested bacteria by quenching fluorescein isothiocyanate-labeled extracellular bacteria with ethidium bromide. This further allowed the exclusion of dead, highly permeable, and subsequently bright-red fluorescent PMNs. Our experiments with two different bacteria, various PMN-to-bacteria ratios (1 : 1, 1 : 10, 1 : 100), and different individuals proved that 1) flow cytometric analysis is accurate and useful for characterizing phagocytosis, 2) adherent bacteria can be distinguished from ingested bacteria after quenching with ethidium bromide, and that 3) phagocytosis kinetics of two bacteria with different onsets of phagocytosis can be determined by flow cytometry and the assessment of a score that quantifies phagocytosis.

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