- Email: [email protected]
Assessment of phagocytic activity of granulocytes using laser flow cytometry
Journal of Immunological Methods, 124 (1989) 231-234
231
Elsevier JIM05362
Assessment of phagocytic activity of granulocytes using laser flow cytometry H. Buschmann and M. Winter Institute for Medical Microbiology and Infectious Medicine, Section of Immunology, VeterinaryFaculty, Universityof Munich, Munich, F..R.G. (Received6 February1989, revised30 May 1989,accepted6 July 1989)
A laser flow cytometer assay for assessing two functions of polymorphonuclear leucocytes is described. The technique permits the quantitation of phagocytosis and intracellular killing of yeast cells by leucocytes and is illustrated with results obtained using porcine polymorphonuclear leucocytes. Key words: Phagocytosis;Granulocyte;Flowcytometry
Introduction
An efficient method for measuring the ingestion and intracellular death of phagocytosed yeast cells by leucocytes has been described by Lehrer and Cline (1969). We modified this assay using laser flow cytometry and in a two-step procedure the number of yeast cells engulfed by the granulocytes and the percentage of yeast cells killed after phagocytosis can be evaluated. After the introduction of several modifications, an efficient but rapid assay was developed which permitted the evaluation of a large number of samples with a high degree of precision.
Materials and methods Granulocyte preparations
Granulocytes were obtained from 20 ml heparinized (25 IU/ml) pig blood after equilibrium density centrifugation on Ficoll-Paque
Correspondence to: H. Buschmarm,Veterinarstrasse 13, D-
8000 Munich22, F.R.G.
(B~Syum, 1968). After centrifugation (30 min, 400 × g) it was possible to obtain granulocytes from the button and lymphocytes from the interface for use in other immunological studies. The cell button was first washed with PBS, pH 7.2, and then twice lysed with 20 ml of cold lysing reagent (8.26 g NH4C1, 1.09 g NaHCO 3, 0.037 g Na-EDTA, made up to 1000 ml with aqua dest.) for 6 rain using slight agitation. After centrifugation (6 rain, 400 x g) and washing twice with PBS the cells were suspended in Hanks' BSS at a concentration of 1 and 5 x 106/ml (purity about 95% granulocytes). Other methods for obtaining granulocytes, for instance by sedimentation on 6% dextran T-70 solution were also used and gave comparable resuits. Baker's yeast was commercially obtained. The viability of the freshly prepared cell suspension, checked by the trypan blue exclusion method, was about 98%. Assessment of the number of yeast cells engulfed by the granulocytes
Baker's yeast was suspended in 10 ml PBS and incubated with 10 ml FITC-PBS (2 mg/ml) for 1 h at room temperature on a magnetic stirrer. After
0022-1759/89/$03.50 © 1989 ElsevierSciencePublishersB.V. (BiomedicalDivision)
232
a further incubation (3 h, 4°C) the yeast cells were washed with PBS (10 rain, 500 x g, 4°C) until the supernatant was free from FITC (determined in a spectrophotometer at 495 nm). After resuspension in PBS the cells were counted by fluorescence microscopy and the homogeneity of the fluorescent labelling of the cells was assessed. Samples with 5 × 106 FITC-labelled yeast cells/ml were frozen ( - 2 0 ° C ) and stored for about 2 weeks. Into 1.5 ml test tubes were pipetted: 100/~1 FITC-labelled yeast cells (5 × 106/113.1), 100/~1 autologous porcine plasma, 200/xl Hanks' BSS, 100 /~1 granulocytes (1 × 106/ml). After 1 h incubation (37 ° C) with continuous agitation (120 rpm) 1 ml iCecold PBS (without Ca 2+ and Mg 2+) was added. The number of granulocytes showing green fluorescence (i.e, phagocytosing cells) was evaluated in an Ortho Spectrum III flow cytometer. The optical specifications: Argon-ion laser, 488 nm emission wavelength, 20 mW operating power, FITC filter (75% transmission between 515 and 530 nm). All other adjustments of the system had been performed according to part no. 168-0012-004 of the Operator's Manual for the Ortho Spectrum III (September 1982). The trigger region was set around the granulocyte cluster (right area in Fig. 1) in a cytogram simultaneously evaluating right angle and forward angle light scattering properties of the cells passing through the focused laser beam. By processing unstained yeast cells the negative region (region A) in the histogram was determined. From this, the first peak of the stained samples could be easily distinguished (Fig. 2). The boundary between region A (negative region) and region B (positive cells) was set so that only 0.5-1.5% of the unstained cells registered in the latter (region B). Alternatively, FITC-labelled polystyrene latex particles (1 btm, Polysciences, Warrington) were processed instead of yeast cells. A quantitative evaluation was performed with the help of a histogram, which showed the number of cells at each level of fluorescent intensity. The relative frequency of cells at different levels of fluorescence intensity was evaluated by determining regions under the peaks observed. In a typical experiment with FITC-labelled yeast cells 25% of the granulocytes were associated with the first peak of specific fluorescence inten-
sity and 75% of the cells were associated with the following regions of higher fluorescence intensity.
Assessment of the proportion of yeast cells killed after phagocytosis by granulocytes Into 1.5 ml test tubes were pipetted: Test
Control
100/~1 100 #1 100/tl
100/~1 100 ~tl 200/~1
100/~l
-
Yeast cell suspension (1 × 106/ml)
Autologousporcine plasma Hanks' BSS Granulocytesuspension (1 X106/ml)
After incubation for 1 h at 37 ° C in a waterbath with continuous agitation 100 /tl cold 2.5% sodium deoxycholate solution (pH 8.7) were added to lyse the granulocytes. To remove DNA released from the lysed granulocytes, 1 ml of a warm (37 ° C) DNAse solution (2 mg deoxyribonuclease I from bovine pancreas = 4000 Kunitz units dissolved in 100 ml PBS) were added and incubated for 5 min with periodic agitation. The tubes were centrifuged (500 × g, 5 min) and the cell pellet resuspended in 1 ml PBS. Then 20 #1 propidium iodide (0.05 m g / m l PBS, Sigma) were added, the tubes incubated for 5 min at 4 ° C, and the percentage of yeast cells showing red fluorescence (i.e., killed cells) evaluated in an Ortho Spectrum III flow cytometer. The optical specifications and all other adjustments were the same as above except that a red pass filter giving 50% transmission at 630 nm was used instead of the FITC filter. The trigger region was set around the yeast cell cluster. The total count (total) represented the total number of yeast cells counted within the trigger region. Histograms of red fluorescence of unstained (region A) and stained (region B) yeast cells were evaluated. Again the boundary between regions A and B was set so that 0.5-1.5% of the unstained cells registered in the latter. To calculate the percentage of yeast cells killed in the sample being analyzed the following formula was used: B%total of test sample-B% total of control = %yeast cells killed
233
In a typical experiment the region B of duplicate test samples gave values of 7357 (55.3%) and 6772 (56.5%); the region B of the complementary controls gave values of 337 (3.3%) and 348 (3.3%). The total counts in the four assays were 13293, 11 986, 10323, and 10578 cells.
12.5 _~
I O0
U
50 E Results and discussion
z
The quantitation of both particle uptake and subsequent killing of ingested particles, was necessary to give a comprehensive estimate of the phagocytic capacity of the granulocytes. This could be done simply by using yeast cells and evaluating by cytofluorometry. For the estimation of the average number of yeast cells ingested by the granulocytes it was necessary for the operator to locate a trigger region in the cytogram (Fig. 1). It can be seen that it was easily possible to differentiate between granulocytes (right spot in Fig. 1) and nonphagocytosed yeast cells (left spot). If the cells within the granulocyte region were analyzed in a histogram showing the fluorescence due to the ingested yeast cells, peaks corresponding to the different numbers of engulfed yeast cells were observed (Fig. 2). When FITC-labelled latex par-
2.5
,a
:-Green
I I B fluorescence
I Intensity
Fig. 2. Histogram showing the number of granulocytes (y axis) after phagocytosis of FITC yeast cells showing different levels of fluorescence intensity (x axis). Regions A (negative) and B (positive) are indicated.
ticles were used in this assay the peaks observed were more distinct (Fig. 3) probably due to the homogeneity of these particles (standard deviation 3% of diameter) and to the equal fluorescent labelling of the particles. The standard deviation of the diameters of the fluorescent yeast cells, however, amounted to 20% of the diameter. Killing of yeast cells after phagocytosis was evaluated following lysis of the neutrophils. Added propidium iodide penetrated the killed yeast cells and stained the DNA to give a red fluorescence (Visser and Van den Engh, 1982). It is important that free DNA is destroyed by DNAse treatment before staining 375 300
tO O
U
225
2 tO
Jo E
LL
Z
150 75
: : : : : : : : : ¢¢,¢,*¢.¢.¢*+¢.t-~c,t-¢4 ¢ , ¢ - i ~ < ~ . ~ . ~
i
I
Right angle
i
i
scatter
Fig. 1. Cytogram differentiating granulocytes (right area) from yeast cells (left area) based on forward scatter and right angle scatter.
Green fluorescence I n t e n s i t y
Fig. 3. Histogram showing the number of granulocytes (y axis) after phagocytosis of FITC latex particles showing different levels of fluorescence intensity (x axis). Regions A (negative) and B (positive) are indicated.
234 with propidium iodide. In addition this treatment avoids clumping of the yeast cells. In pilot studies it had been shown that no significant alteration of the yeast cells occurred even after treatment with DNAse for 25 n-fin. Using this assay the percentage of killed yeast cells could be evaluated from a cytofluorographic histogram with a high degree of accuracy. On average more than 10 000 yeast cells were evaluated by the cytofluorograph in each run. Control values (samples without granulocytes) were always substracted from the test values. A mean of about 40% of the yeast cells stained red after a 1 h incubation. The values from individual pigs, however, showed great variation, thus demonstrating differences in the phagocytic capacity between individuals (mean killing rate from 77 clinically healthy G e r m a n Landrace pigs: 42.19 _+ 8.11%). When samples from 30 pigs were evaluated according to the original method of Lehrer and Cline (1969) the killing rate of the yeast cells was 40.18 +_ 13.95%; 300 yeast cells were evaluated in each smear under the microscope. Using laser flow cytometry the subjective and tedious aspects of microscopic evaluation can be avoided and the precision of the measurements is considerably increased. In their studies Lehrer and Cline (1969) showed that all added Candida albicans yeast cells had been ingested by the neutrophils after 15 min incubation. Candidacidal activity was independent of the Candida:neutrophil ratio over a range of 0.5-1.5. Candida cells per neutrophil. F r o m these observations and taking into consideration the incubation time of 1 h used in the present assay we conclude that few if any uningested yeast cells will remain at the end.
In further studies with pig granulocytes (Winter and Buschmann, 1987) using the assay of Lehrer and Chne (1969) yeast cell killing was positively correlated (r = 0.37) with the glucose-1-14C oxidation rate (respiratory burst) during phagocytosis of killed Staphylococcus epidermidis (Keusch et al., 1973). There was no correlation, however, with the rate of in vitro reduction of nitroblue tetrazolium (NBT) to insoluble formazan (r = - 0 . 1 2 ) (Baehner et al., 1976) or with luminol enhanced chemihiminescence of granulocytes produced by phorbol-myristate-acetate (PMA) (r = - 0 . 1 4 ) (De Chatelet and Shirley, 1981).
References Baehner, R.L., Boxer, L.A. and Davis, J. (1976) The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leucocytes. Blood 48, 309. B~yum, A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21, 1. De Chatelet, C.R. and Shirley, P.S. (1981) Evaluation of chronic granulomatous disease by a chemiluminescent assay of microlitre quantities of whole blood. Clin. Chem. 27, 1739. Keusch, G.T., Douglas, S.D., Mildvan, D. and Hirschman, S.Z. (1973) 14C-glucose oxidation in whole blood: a clinical assay for phagocyte dysfunction. Infect. Immun. 5, 414. Lehrer, R.I. and Cline, M.J. (1969) Interaction of Candida albicans with human leucocytes and serum. J. Bacteriol. 98, 996. Visser, J.W.M. and Van den Engh, G.J. (1982) Immunofluorescence measurements by flow cytometry. In: G. Wick, K.N. Traill and K. Schauenstein (Eds.), Immunofluorescence Technology. Selected Theoretical and Clinical Aspects. Amsterdam, Ch. 7. Winter, M. and Buschmann, H.G. (1987) Measuring phagocytic capacity in polymorphonuclear cells of the pig - a comparison between different assays. J. Vet. Med. B34, 504.
231
Elsevier JIM05362
Assessment of phagocytic activity of granulocytes using laser flow cytometry H. Buschmann and M. Winter Institute for Medical Microbiology and Infectious Medicine, Section of Immunology, VeterinaryFaculty, Universityof Munich, Munich, F..R.G. (Received6 February1989, revised30 May 1989,accepted6 July 1989)
A laser flow cytometer assay for assessing two functions of polymorphonuclear leucocytes is described. The technique permits the quantitation of phagocytosis and intracellular killing of yeast cells by leucocytes and is illustrated with results obtained using porcine polymorphonuclear leucocytes. Key words: Phagocytosis;Granulocyte;Flowcytometry
Introduction
An efficient method for measuring the ingestion and intracellular death of phagocytosed yeast cells by leucocytes has been described by Lehrer and Cline (1969). We modified this assay using laser flow cytometry and in a two-step procedure the number of yeast cells engulfed by the granulocytes and the percentage of yeast cells killed after phagocytosis can be evaluated. After the introduction of several modifications, an efficient but rapid assay was developed which permitted the evaluation of a large number of samples with a high degree of precision.
Materials and methods Granulocyte preparations
Granulocytes were obtained from 20 ml heparinized (25 IU/ml) pig blood after equilibrium density centrifugation on Ficoll-Paque
Correspondence to: H. Buschmarm,Veterinarstrasse 13, D-
8000 Munich22, F.R.G.
(B~Syum, 1968). After centrifugation (30 min, 400 × g) it was possible to obtain granulocytes from the button and lymphocytes from the interface for use in other immunological studies. The cell button was first washed with PBS, pH 7.2, and then twice lysed with 20 ml of cold lysing reagent (8.26 g NH4C1, 1.09 g NaHCO 3, 0.037 g Na-EDTA, made up to 1000 ml with aqua dest.) for 6 rain using slight agitation. After centrifugation (6 rain, 400 x g) and washing twice with PBS the cells were suspended in Hanks' BSS at a concentration of 1 and 5 x 106/ml (purity about 95% granulocytes). Other methods for obtaining granulocytes, for instance by sedimentation on 6% dextran T-70 solution were also used and gave comparable resuits. Baker's yeast was commercially obtained. The viability of the freshly prepared cell suspension, checked by the trypan blue exclusion method, was about 98%. Assessment of the number of yeast cells engulfed by the granulocytes
Baker's yeast was suspended in 10 ml PBS and incubated with 10 ml FITC-PBS (2 mg/ml) for 1 h at room temperature on a magnetic stirrer. After
0022-1759/89/$03.50 © 1989 ElsevierSciencePublishersB.V. (BiomedicalDivision)
232
a further incubation (3 h, 4°C) the yeast cells were washed with PBS (10 rain, 500 x g, 4°C) until the supernatant was free from FITC (determined in a spectrophotometer at 495 nm). After resuspension in PBS the cells were counted by fluorescence microscopy and the homogeneity of the fluorescent labelling of the cells was assessed. Samples with 5 × 106 FITC-labelled yeast cells/ml were frozen ( - 2 0 ° C ) and stored for about 2 weeks. Into 1.5 ml test tubes were pipetted: 100/~1 FITC-labelled yeast cells (5 × 106/113.1), 100/~1 autologous porcine plasma, 200/xl Hanks' BSS, 100 /~1 granulocytes (1 × 106/ml). After 1 h incubation (37 ° C) with continuous agitation (120 rpm) 1 ml iCecold PBS (without Ca 2+ and Mg 2+) was added. The number of granulocytes showing green fluorescence (i.e, phagocytosing cells) was evaluated in an Ortho Spectrum III flow cytometer. The optical specifications: Argon-ion laser, 488 nm emission wavelength, 20 mW operating power, FITC filter (75% transmission between 515 and 530 nm). All other adjustments of the system had been performed according to part no. 168-0012-004 of the Operator's Manual for the Ortho Spectrum III (September 1982). The trigger region was set around the granulocyte cluster (right area in Fig. 1) in a cytogram simultaneously evaluating right angle and forward angle light scattering properties of the cells passing through the focused laser beam. By processing unstained yeast cells the negative region (region A) in the histogram was determined. From this, the first peak of the stained samples could be easily distinguished (Fig. 2). The boundary between region A (negative region) and region B (positive cells) was set so that only 0.5-1.5% of the unstained cells registered in the latter (region B). Alternatively, FITC-labelled polystyrene latex particles (1 btm, Polysciences, Warrington) were processed instead of yeast cells. A quantitative evaluation was performed with the help of a histogram, which showed the number of cells at each level of fluorescent intensity. The relative frequency of cells at different levels of fluorescence intensity was evaluated by determining regions under the peaks observed. In a typical experiment with FITC-labelled yeast cells 25% of the granulocytes were associated with the first peak of specific fluorescence inten-
sity and 75% of the cells were associated with the following regions of higher fluorescence intensity.
Assessment of the proportion of yeast cells killed after phagocytosis by granulocytes Into 1.5 ml test tubes were pipetted: Test
Control
100/~1 100 #1 100/tl
100/~1 100 ~tl 200/~1
100/~l
-
Yeast cell suspension (1 × 106/ml)
Autologousporcine plasma Hanks' BSS Granulocytesuspension (1 X106/ml)
After incubation for 1 h at 37 ° C in a waterbath with continuous agitation 100 /tl cold 2.5% sodium deoxycholate solution (pH 8.7) were added to lyse the granulocytes. To remove DNA released from the lysed granulocytes, 1 ml of a warm (37 ° C) DNAse solution (2 mg deoxyribonuclease I from bovine pancreas = 4000 Kunitz units dissolved in 100 ml PBS) were added and incubated for 5 min with periodic agitation. The tubes were centrifuged (500 × g, 5 min) and the cell pellet resuspended in 1 ml PBS. Then 20 #1 propidium iodide (0.05 m g / m l PBS, Sigma) were added, the tubes incubated for 5 min at 4 ° C, and the percentage of yeast cells showing red fluorescence (i.e., killed cells) evaluated in an Ortho Spectrum III flow cytometer. The optical specifications and all other adjustments were the same as above except that a red pass filter giving 50% transmission at 630 nm was used instead of the FITC filter. The trigger region was set around the yeast cell cluster. The total count (total) represented the total number of yeast cells counted within the trigger region. Histograms of red fluorescence of unstained (region A) and stained (region B) yeast cells were evaluated. Again the boundary between regions A and B was set so that 0.5-1.5% of the unstained cells registered in the latter. To calculate the percentage of yeast cells killed in the sample being analyzed the following formula was used: B%total of test sample-B% total of control = %yeast cells killed
233
In a typical experiment the region B of duplicate test samples gave values of 7357 (55.3%) and 6772 (56.5%); the region B of the complementary controls gave values of 337 (3.3%) and 348 (3.3%). The total counts in the four assays were 13293, 11 986, 10323, and 10578 cells.
12.5 _~
I O0
U
50 E Results and discussion
z
The quantitation of both particle uptake and subsequent killing of ingested particles, was necessary to give a comprehensive estimate of the phagocytic capacity of the granulocytes. This could be done simply by using yeast cells and evaluating by cytofluorometry. For the estimation of the average number of yeast cells ingested by the granulocytes it was necessary for the operator to locate a trigger region in the cytogram (Fig. 1). It can be seen that it was easily possible to differentiate between granulocytes (right spot in Fig. 1) and nonphagocytosed yeast cells (left spot). If the cells within the granulocyte region were analyzed in a histogram showing the fluorescence due to the ingested yeast cells, peaks corresponding to the different numbers of engulfed yeast cells were observed (Fig. 2). When FITC-labelled latex par-
2.5
,a
:-Green
I I B fluorescence
I Intensity
Fig. 2. Histogram showing the number of granulocytes (y axis) after phagocytosis of FITC yeast cells showing different levels of fluorescence intensity (x axis). Regions A (negative) and B (positive) are indicated.
ticles were used in this assay the peaks observed were more distinct (Fig. 3) probably due to the homogeneity of these particles (standard deviation 3% of diameter) and to the equal fluorescent labelling of the particles. The standard deviation of the diameters of the fluorescent yeast cells, however, amounted to 20% of the diameter. Killing of yeast cells after phagocytosis was evaluated following lysis of the neutrophils. Added propidium iodide penetrated the killed yeast cells and stained the DNA to give a red fluorescence (Visser and Van den Engh, 1982). It is important that free DNA is destroyed by DNAse treatment before staining 375 300
tO O
U
225
2 tO
Jo E
LL
Z
150 75
: : : : : : : : : ¢¢,¢,*¢.¢.¢*+¢.t-~c,t-¢4 ¢ , ¢ - i ~ < ~ . ~ . ~
i
I
Right angle
i
i
scatter
Fig. 1. Cytogram differentiating granulocytes (right area) from yeast cells (left area) based on forward scatter and right angle scatter.
Green fluorescence I n t e n s i t y
Fig. 3. Histogram showing the number of granulocytes (y axis) after phagocytosis of FITC latex particles showing different levels of fluorescence intensity (x axis). Regions A (negative) and B (positive) are indicated.
234 with propidium iodide. In addition this treatment avoids clumping of the yeast cells. In pilot studies it had been shown that no significant alteration of the yeast cells occurred even after treatment with DNAse for 25 n-fin. Using this assay the percentage of killed yeast cells could be evaluated from a cytofluorographic histogram with a high degree of accuracy. On average more than 10 000 yeast cells were evaluated by the cytofluorograph in each run. Control values (samples without granulocytes) were always substracted from the test values. A mean of about 40% of the yeast cells stained red after a 1 h incubation. The values from individual pigs, however, showed great variation, thus demonstrating differences in the phagocytic capacity between individuals (mean killing rate from 77 clinically healthy G e r m a n Landrace pigs: 42.19 _+ 8.11%). When samples from 30 pigs were evaluated according to the original method of Lehrer and Cline (1969) the killing rate of the yeast cells was 40.18 +_ 13.95%; 300 yeast cells were evaluated in each smear under the microscope. Using laser flow cytometry the subjective and tedious aspects of microscopic evaluation can be avoided and the precision of the measurements is considerably increased. In their studies Lehrer and Cline (1969) showed that all added Candida albicans yeast cells had been ingested by the neutrophils after 15 min incubation. Candidacidal activity was independent of the Candida:neutrophil ratio over a range of 0.5-1.5. Candida cells per neutrophil. F r o m these observations and taking into consideration the incubation time of 1 h used in the present assay we conclude that few if any uningested yeast cells will remain at the end.
In further studies with pig granulocytes (Winter and Buschmann, 1987) using the assay of Lehrer and Chne (1969) yeast cell killing was positively correlated (r = 0.37) with the glucose-1-14C oxidation rate (respiratory burst) during phagocytosis of killed Staphylococcus epidermidis (Keusch et al., 1973). There was no correlation, however, with the rate of in vitro reduction of nitroblue tetrazolium (NBT) to insoluble formazan (r = - 0 . 1 2 ) (Baehner et al., 1976) or with luminol enhanced chemihiminescence of granulocytes produced by phorbol-myristate-acetate (PMA) (r = - 0 . 1 4 ) (De Chatelet and Shirley, 1981).
References Baehner, R.L., Boxer, L.A. and Davis, J. (1976) The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leucocytes. Blood 48, 309. B~yum, A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. Clin. Lab. Invest. 21, 1. De Chatelet, C.R. and Shirley, P.S. (1981) Evaluation of chronic granulomatous disease by a chemiluminescent assay of microlitre quantities of whole blood. Clin. Chem. 27, 1739. Keusch, G.T., Douglas, S.D., Mildvan, D. and Hirschman, S.Z. (1973) 14C-glucose oxidation in whole blood: a clinical assay for phagocyte dysfunction. Infect. Immun. 5, 414. Lehrer, R.I. and Cline, M.J. (1969) Interaction of Candida albicans with human leucocytes and serum. J. Bacteriol. 98, 996. Visser, J.W.M. and Van den Engh, G.J. (1982) Immunofluorescence measurements by flow cytometry. In: G. Wick, K.N. Traill and K. Schauenstein (Eds.), Immunofluorescence Technology. Selected Theoretical and Clinical Aspects. Amsterdam, Ch. 7. Winter, M. and Buschmann, H.G. (1987) Measuring phagocytic capacity in polymorphonuclear cells of the pig - a comparison between different assays. J. Vet. Med. B34, 504.