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Rapid, single-step isolation of equine neutrophils on a discontinuous Percoll density gradient
SHORT COMMUNICATIONS Research in Veterinary Science 1987,42, 4JJ-412
Rapid, single-step isolation of equine neutrophils on a density gradient
disc~,..tinuous
Percoll
J. F. PYCOCK, W. EDWARD ALLEN, T. H. MORRIS, Department of Surgery and Obstetrics, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA
A rapid single-step procedure for isolation of equine neutrophils (PMNS) from peripheral blood is described. A discontinuous gradient of two Percoll solutions (densities of ],087 kg litre-I and ]']08 kg litre-I was used. The PMNS were isolated to more than 95 per cent purity with a viability of more than 99 per cent and a cell recovery of more than 83 per cent. The method used was rapid and reproducible and the equipment required is relatively simple. The function of the recovered cells was assessed in a chemotactic assay using a modified Boyden chamber technique. THE ideal method of isolating equine neutrophils (PMNS) should be rapid and cause minimal damage to ensure cell viability which is essential for functional evaluation. Sedimentation of erythrocytes is commonly used to produce a leucocyte rich supernatant. The mononuclear cells and the granulocytes are then separated by differential or density gradient centrifugation (Babior and Cohen 1981). In man sedimentation is usually enhanced by adding Dextran to encourage the aggregation of erythrocytes, but the very rapid erythrocyte sedimentation rate in the horse (Richter 1966) makes this unnecessary. Newer methods of isolation involve single step density gradient centrifugation. Initially these were Ficoll-Hypaque solutions (Ferrante and Thong 1980) but more recently Percoll gradients have been used (Hjorth et aI19l:S1, Jepsen and Skottun 1982, Harbeck et al 1982). This paper describes the development and use of such a method for the isolation of equine PMNS. Blood samples (n = 31) were collected from 15 clinically normal horses and 16 ponies by jugular venepuncture into 5 ml syringes containing ethylene diamine tetra-acetic acid (Monovette; Sarstedt) and stored on ice. Stock iso-osmotic Perc 011 solution was prepared by adding nine volumes of commercial Percoll (density 1·128 kg litre - I; Pharmacia Fine Chemicals), to one volume of 10 x Hank's balanced salt solution (HBSS; Flow Laboratories). Further dilutions were made using I x HBSS, to achieve densities based on the calibration curve supplied by the manufacturer. The discontinuous gradient was produced by placing 2·5 mlof70percent Percollsolution(I'087kglitre- l )intoa 14 ml clear, conical, polystyrene centrifuge tube (Henley's Medical Supplies) using a sterile 5 ml syringe and 0·9 x 50 mm needle, with 2· 5 ml of 85 per cent Perc 011 (I . 108 kg litre - J) carefully layered underneath, avoiding any mixing of the two layers. Blood (3 ml) was then layered on to the top of the gradient. The tubes were centrifuged at 400 g for 20 minutes at room temperature (Super MUltex Centrifuge Mark Three; Measuring and Scientific Equipment). The centrifuge was not braked as this was found to disrupt the final gradient.
The result was a gradient where the mononuclear cells were layered above the less dense Percoll solution, PMNS were layered between the two Percoll solutions and the erythrocytes and eosinophils sedimented to the bottom (Fig I). Leucocytes were harvested by removing the plasma with a pasteur pipette and then aspirating the cell layer with a fine tip Pastette (Alpha Laboratories). This was transferred to a second centrifuge tube containing 2 ml of I x HBSS. The resulting suspension was centrifuged at 200 g for eight minutes, the supernatant discarded and the cell pellet resuspended in 2 ml of I x HBSS. The cells were then counted on a ZM Coulter Counter (Coulter Electronics). Differential cell counts were made manually, before and after Perc 011 separation by making smears on microscope slides and staining them with M + D Diff-Kwik (American Hospital Supplies, Didcot). A total of 100 cells were counted and the percentages of PMNS calculated. Viability of 100 cells was assessed by Trypan blue exclusion (Babior and Cohen 1981). Leucocyte locomotion was assessed in a modified Boyden Chamber using Zymosan-activated plasma in the lower chamber (Sedgwick et al 1987).
411
[PlaSma
-_[Le'COC'le,
J
Pe,coll
Tube markings
. -Leucocytes
Perc 011
Erythrocytes
FIG 1: Perc011 gradient after centrifugation . 'Tube markings' refers to moulding marks in the plastic tube
J. F. Pycock, W. Edward Allen, T. H. Morris
412 TABLE 1: Isolated cell fraction
Purity ISE)
%PMN
Pony (n ~ 16) Horse In ~ 15)
98·1 97·6
(0·55) 10·76)
Recovery ISE)
%PMN
83·9 86·8
(2'9) (2·8)
Viability (SE)
%PMN
99·9 99·2
(0,13) (0,26)
PMN Equine neutrophils
SE Standard error
Percoll has previously been used to isolate equine PMNS, (Jacobsen et al 1982), but the method described involved several steps and took three hours to complete. Dextran has been added to equine blood to enhance erythrocyte sedimentation (Lui et al 1985) but using human blood, Hjorth et al (1981) obtained a greater yield, purity and viability of PMNS using Percoll than Dextran sedimentation. The advantage of Percoll over other density gradient media that have been used in the horse, such as Ficoll-Hypaque (Coignoul et al 1983) is that it is iso-osmotic and its viscosity is lower so that centrifugation times are shorter and cell trauma is minimised. Differences in Percoll density as used at room temperature in this study and those of Sedgwick et al (1986), who used a refrigerated centrifuge, are explained by the effect of temperature on Percoil density (Jepsen and Skottun 1982). The lack of contamination of the PMN fraction by erythrocytes is probably caused by the rapid sedimentation rate in the horse (Richter 1966) and precludes the use of a lysis procedure. The viability of the PMNS isolated in this study (99 per cent) compares well with a viability of 88 per cent using FicollHypaque (Bertram and Coignoul 1982). This high viability assessed using Trypan blue exclusion was confirmed using the Boyden chamber technique. The results of 31 blood samples treated in this way are shown in Table I. Using the above method of isolation assessments of PMN locomotion were made using the Boyden chamber technique. Using the leading front counting method (Zigmond and Hirsch 1973) PMNS moved 147 /Am (SE 3· 58, n = I I) when Zymosanactivated plasma was the chemoattractant in the lower chamber as compared to 45 /Am (SE 2· 67, n = I I) in response to HBSS. As purity and recovery were also good the method described in this study could also be used to isolate the mononuclear leucocytes in the upper cell band. Eosinophils
sediment to the bottom of the tube with the erythrocytes (Riding and Willadsen 1981) and these cells could be recovered by a lysis procedure. The method described is simple, rapid and reproducible and the isolated cells appear to function normally in studies of leucocyte locomotion. Acknowledgements The authors are grateful to Mr D. Gunn and Miss A. Jackman for technical assistance and to the Wellcome Trust and the Agricultural and Food Research Council for financial support. References BABIOR, B. M. & COHEN, H. J. (1981) Leucocyte Function. Ed M. J. Cline. London, Churchill Livingstone. p I BERTRAM, T. A. & COIGNOUL, F. L. (1982) Veterinary Pathology 19, 534-543 COIGNOUL, F. L., BERTRAM, T. A., ROTH, J. A. & CHEVILLE, N. F. (1983) American Journal of Veterinary Research 45, 898-902 FERRANTE, A. & THONG, Y. H. (1980) Journal of Immunological Methods 36, 109- I 17 HARBECK, R. J., HOFFMAN, A. A., REDECKER, 5., BIUNDO, T. & KURNICK, J. (1982) Clinical Immunology and Immunopathology 23, 682-690 HJORTH, R., JONSSON, A.-K. & VRETLAB, P. A. (1981) Journal of Immunological Methods 43,95-101 JACOBSEN, K., GINTZ, T., REED, S. M., NEWBRY, J., BAYLY, W. M., PERRYMAN, L. E. & LEID, R. W. (1982) American Journal of Veterinary Research 43,1912-1916 JEPSEN, L. V. & SKOTTUN, T. (1982) Scandinavian Journal of Clinical Laboratory Investigation 42, 235-238 LUI, I. K. M., CHEUNG, A. T. W., WALSH, E. M., MILLER, M. E. & LINDENBERG, P. M. (1985) American Journal of Veterinary Research 49,917-920 RICHTER, W. (1966) Acta Chirurgica Scandinavica 132, 601-612 RIDING, G. A. & WILLADSEN, P. (1981) Journal of Immunological Methods 46, 113-119 SEDGWICK, A. D., DAWSON, J. & LEES, P. (1987) Research in Veterinary Science (In press) SEDGWICK, A. D., MORRIS, T. H., RUSSELL, B. A. & LEES, P. (1986) Veterinary Research Communications 10, 445-452 ZIGMOND, S. H. & HIRSCH, J. G. (1973) Journal of Experimental Medicine 137, 387-410
Received for publication August 20, 1986 Accepted December 8, 1986
Rapid, single-step isolation of equine neutrophils on a density gradient
disc~,..tinuous
Percoll
J. F. PYCOCK, W. EDWARD ALLEN, T. H. MORRIS, Department of Surgery and Obstetrics, Royal Veterinary College, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA
A rapid single-step procedure for isolation of equine neutrophils (PMNS) from peripheral blood is described. A discontinuous gradient of two Percoll solutions (densities of ],087 kg litre-I and ]']08 kg litre-I was used. The PMNS were isolated to more than 95 per cent purity with a viability of more than 99 per cent and a cell recovery of more than 83 per cent. The method used was rapid and reproducible and the equipment required is relatively simple. The function of the recovered cells was assessed in a chemotactic assay using a modified Boyden chamber technique. THE ideal method of isolating equine neutrophils (PMNS) should be rapid and cause minimal damage to ensure cell viability which is essential for functional evaluation. Sedimentation of erythrocytes is commonly used to produce a leucocyte rich supernatant. The mononuclear cells and the granulocytes are then separated by differential or density gradient centrifugation (Babior and Cohen 1981). In man sedimentation is usually enhanced by adding Dextran to encourage the aggregation of erythrocytes, but the very rapid erythrocyte sedimentation rate in the horse (Richter 1966) makes this unnecessary. Newer methods of isolation involve single step density gradient centrifugation. Initially these were Ficoll-Hypaque solutions (Ferrante and Thong 1980) but more recently Percoll gradients have been used (Hjorth et aI19l:S1, Jepsen and Skottun 1982, Harbeck et al 1982). This paper describes the development and use of such a method for the isolation of equine PMNS. Blood samples (n = 31) were collected from 15 clinically normal horses and 16 ponies by jugular venepuncture into 5 ml syringes containing ethylene diamine tetra-acetic acid (Monovette; Sarstedt) and stored on ice. Stock iso-osmotic Perc 011 solution was prepared by adding nine volumes of commercial Percoll (density 1·128 kg litre - I; Pharmacia Fine Chemicals), to one volume of 10 x Hank's balanced salt solution (HBSS; Flow Laboratories). Further dilutions were made using I x HBSS, to achieve densities based on the calibration curve supplied by the manufacturer. The discontinuous gradient was produced by placing 2·5 mlof70percent Percollsolution(I'087kglitre- l )intoa 14 ml clear, conical, polystyrene centrifuge tube (Henley's Medical Supplies) using a sterile 5 ml syringe and 0·9 x 50 mm needle, with 2· 5 ml of 85 per cent Perc 011 (I . 108 kg litre - J) carefully layered underneath, avoiding any mixing of the two layers. Blood (3 ml) was then layered on to the top of the gradient. The tubes were centrifuged at 400 g for 20 minutes at room temperature (Super MUltex Centrifuge Mark Three; Measuring and Scientific Equipment). The centrifuge was not braked as this was found to disrupt the final gradient.
The result was a gradient where the mononuclear cells were layered above the less dense Percoll solution, PMNS were layered between the two Percoll solutions and the erythrocytes and eosinophils sedimented to the bottom (Fig I). Leucocytes were harvested by removing the plasma with a pasteur pipette and then aspirating the cell layer with a fine tip Pastette (Alpha Laboratories). This was transferred to a second centrifuge tube containing 2 ml of I x HBSS. The resulting suspension was centrifuged at 200 g for eight minutes, the supernatant discarded and the cell pellet resuspended in 2 ml of I x HBSS. The cells were then counted on a ZM Coulter Counter (Coulter Electronics). Differential cell counts were made manually, before and after Perc 011 separation by making smears on microscope slides and staining them with M + D Diff-Kwik (American Hospital Supplies, Didcot). A total of 100 cells were counted and the percentages of PMNS calculated. Viability of 100 cells was assessed by Trypan blue exclusion (Babior and Cohen 1981). Leucocyte locomotion was assessed in a modified Boyden Chamber using Zymosan-activated plasma in the lower chamber (Sedgwick et al 1987).
411
[PlaSma
-_[Le'COC'le,
J
Pe,coll
Tube markings
. -Leucocytes
Perc 011
Erythrocytes
FIG 1: Perc011 gradient after centrifugation . 'Tube markings' refers to moulding marks in the plastic tube
J. F. Pycock, W. Edward Allen, T. H. Morris
412 TABLE 1: Isolated cell fraction
Purity ISE)
%PMN
Pony (n ~ 16) Horse In ~ 15)
98·1 97·6
(0·55) 10·76)
Recovery ISE)
%PMN
83·9 86·8
(2'9) (2·8)
Viability (SE)
%PMN
99·9 99·2
(0,13) (0,26)
PMN Equine neutrophils
SE Standard error
Percoll has previously been used to isolate equine PMNS, (Jacobsen et al 1982), but the method described involved several steps and took three hours to complete. Dextran has been added to equine blood to enhance erythrocyte sedimentation (Lui et al 1985) but using human blood, Hjorth et al (1981) obtained a greater yield, purity and viability of PMNS using Percoll than Dextran sedimentation. The advantage of Percoll over other density gradient media that have been used in the horse, such as Ficoll-Hypaque (Coignoul et al 1983) is that it is iso-osmotic and its viscosity is lower so that centrifugation times are shorter and cell trauma is minimised. Differences in Percoll density as used at room temperature in this study and those of Sedgwick et al (1986), who used a refrigerated centrifuge, are explained by the effect of temperature on Percoil density (Jepsen and Skottun 1982). The lack of contamination of the PMN fraction by erythrocytes is probably caused by the rapid sedimentation rate in the horse (Richter 1966) and precludes the use of a lysis procedure. The viability of the PMNS isolated in this study (99 per cent) compares well with a viability of 88 per cent using FicollHypaque (Bertram and Coignoul 1982). This high viability assessed using Trypan blue exclusion was confirmed using the Boyden chamber technique. The results of 31 blood samples treated in this way are shown in Table I. Using the above method of isolation assessments of PMN locomotion were made using the Boyden chamber technique. Using the leading front counting method (Zigmond and Hirsch 1973) PMNS moved 147 /Am (SE 3· 58, n = I I) when Zymosanactivated plasma was the chemoattractant in the lower chamber as compared to 45 /Am (SE 2· 67, n = I I) in response to HBSS. As purity and recovery were also good the method described in this study could also be used to isolate the mononuclear leucocytes in the upper cell band. Eosinophils
sediment to the bottom of the tube with the erythrocytes (Riding and Willadsen 1981) and these cells could be recovered by a lysis procedure. The method described is simple, rapid and reproducible and the isolated cells appear to function normally in studies of leucocyte locomotion. Acknowledgements The authors are grateful to Mr D. Gunn and Miss A. Jackman for technical assistance and to the Wellcome Trust and the Agricultural and Food Research Council for financial support. References BABIOR, B. M. & COHEN, H. J. (1981) Leucocyte Function. Ed M. J. Cline. London, Churchill Livingstone. p I BERTRAM, T. A. & COIGNOUL, F. L. (1982) Veterinary Pathology 19, 534-543 COIGNOUL, F. L., BERTRAM, T. A., ROTH, J. A. & CHEVILLE, N. F. (1983) American Journal of Veterinary Research 45, 898-902 FERRANTE, A. & THONG, Y. H. (1980) Journal of Immunological Methods 36, 109- I 17 HARBECK, R. J., HOFFMAN, A. A., REDECKER, 5., BIUNDO, T. & KURNICK, J. (1982) Clinical Immunology and Immunopathology 23, 682-690 HJORTH, R., JONSSON, A.-K. & VRETLAB, P. A. (1981) Journal of Immunological Methods 43,95-101 JACOBSEN, K., GINTZ, T., REED, S. M., NEWBRY, J., BAYLY, W. M., PERRYMAN, L. E. & LEID, R. W. (1982) American Journal of Veterinary Research 43,1912-1916 JEPSEN, L. V. & SKOTTUN, T. (1982) Scandinavian Journal of Clinical Laboratory Investigation 42, 235-238 LUI, I. K. M., CHEUNG, A. T. W., WALSH, E. M., MILLER, M. E. & LINDENBERG, P. M. (1985) American Journal of Veterinary Research 49,917-920 RICHTER, W. (1966) Acta Chirurgica Scandinavica 132, 601-612 RIDING, G. A. & WILLADSEN, P. (1981) Journal of Immunological Methods 46, 113-119 SEDGWICK, A. D., DAWSON, J. & LEES, P. (1987) Research in Veterinary Science (In press) SEDGWICK, A. D., MORRIS, T. H., RUSSELL, B. A. & LEES, P. (1986) Veterinary Research Communications 10, 445-452 ZIGMOND, S. H. & HIRSCH, J. G. (1973) Journal of Experimental Medicine 137, 387-410
Received for publication August 20, 1986 Accepted December 8, 1986