Research in Veterinary Science 1991, 50, 116-117
Isolation of equine peripheral blood mononuclear cells using Percoli S. A. MAY*, R. E. H O O K E t , P. LEES, Royal Veterinary College, Hawkshead House, North Mymms,
Hatfield, Hertfordshire AL9 7TA
The concentration of Pereoll required for isolating equine peripheral blood mononuclear cells has been reinvestigated. A poor cell yield was obtained at the 60 per cent concentration already reported. It is recommended that workers specifically interested in high yields of mononuclear cells, for investigation of lymphocyte and monocyte functions, use a concentration of 65 per cent Percoll. However, workers wishing to isolate pure populations of equine neutrophils might consider a concentration of 70 per cent in the upper layer of Percoll used to retain the mononuclear cells.
blood at 4°C gently layered on top of each. The tubes were centrifuged at 440 g for 30 minutes at 4°C, the white cell layer above the Percoll harvested, and total and differential white cell counts performed. The results are displayed in Fig 1. All the mononuclear cells present in the blood were recovered on 65 per cent and 67.5 per cent Percoll, without any neutrophil contamination, compared with 43 per cent of mononuclear cells recovered on 60 per cent Percoll. Although all mononuclear cells were recovered at concentrations above 67.5 per cent, increasing contamination with neutrophils occurred. Therefore, a concentration of 65 per cent Percoll is recommended for extracting mononuclear cells from equine blood. Fig 1 shows that at 4°C a discontinuous gradient of the type recommended by Sedgwick et al (1986) of 60 per cent and 75 per cent Percoll, results in poor recovery of m o n o nuclear cells above the 60 per cent Percoll (although these are pure) and poor recovery of neutrophils, contaminated by the rest of the mononuclear ceils, above the 75 per cent Percoll. The concentration of the upper layer of Percoll must be in the range of 65 to 67.5 per cent if this contamination of the neutrophil layer is to be avoided. Thus, a 65 per cent/75 per cent gradient would yield relatively pure populations of cells, although only 60 per cent of the neutrophils would be recovered on the bottom layer. Another group of workers has described a method for isolating equine neutrophils at room temperature. This involves a discontinuous Percoll gradient of 70 per cent/ 85 per cent (Pycock et a11987). These workers report a purity
IN order to undertake studies on individual cell populations in vitro, it is first necessary to isolate the cells of interest. In the case of blood cells, it is possible to exploit differences in the density of each type of cell to achieve separation of pure populations on continuous or discontinuous density gradients (Pycock et al 1987). The isolation of equine neutrophils and equine mononuclear cells at 4°C on a discontinuous Percoll (Pharmacia) gradient, using 60 per cent Percoll to retain the mononuclear cells and 75 per cent Percoll to retain the neutrophils, has been described, with the authors reporting 99 per cent purity of the mononuclear cell fraction and 94 per cent purity of the neutrophil fraction (Sedgwick et al 1986). However, problems with this technique, involving low yields of mononuclear cells relative to the total numbers present in the original blood, and contamination of the neutrophil fraction with large numbers of mononuclear cells, led us to reinvestigate the optimal density for recovery of the mononuclear cell fraction. The purpose of this communication is to report the density of Percoll required for retention of the whole of the mononuclear cell fraction without significant contamination by neutrophils. The methodology of Sedgwick et al (1986) was retained, except that Hanks' balanced salt solution (HBSS) replaced m i n i m u m essential medium. 'Stock' Percoll was prepared by mixing nine parts Percoll with one part 10 × concentrated HBSS. This stock was diluted further with HBSS, and the concentration expressed as percentage stock Percoll. Thus 6 ml stock Percoll and 4 ml HBSS were designated 60 per cent. A range of Percoll concentrations from 60 to 75 per cent, at 2.5 per cent intervals, was investigated. A volume of 2-5 ml of each concentration of Percoll was placed in centrifuge tubes at 4°C and 2 ml heparinised whole equine
/
*Present address: Division of Equine Studies, University of Liverpool Veterinary Field Station, Leahurst, Neston, Wirral, L64 7TE tPresent address: Duphar Veterinary Ltd, Solvay House, Hedge End, Southampton, SO3 4QH
o~ 60
Q
/ Q ~ , 65 70 Percoll concentration (%)
,
, 75
FIG 1 : The effect of gradient density on white blood cell recovery. • • Total cells, © 0 Neutrophils, ~ A Mononuclear ceils
116
Percoll isolation o f equine b l o o d cells of 98 per cent and a recovery of 84 to 87 per cent. Although, an 85 per cent concentration of Percoll was not investigated in this study, it will be seen by reference to Fig 1 that this is likely to retain all the neutrophils. In addition, it can be seen that the 70 per cent Percoll retains all the mononuclear cells and, in this experiment, about 8 per cent of the neutrophils. This accords with the levels of purity and recovery reported by Pycock et al (1987). Thus, investigators interested mainly in pure populations of neutrophils should choose a slightly higher concentration of Percoll in the upper layer, such as 70 per cent, whereas others wishing to isolate mononuclear cells should choose the concentration of 65 per cent as outlined above. In discussing the difference between their results and those of Sedgwick et al (1986), Pycock et al (1987) cited the effect of temperature on Percoll density (Jepsen and Skottun 1982). However, the results reported here demonstrate that the original concentrations of Sedgwick et al (1986) do not yield the pure populations of cells intended. In addition, to obtain an equivalent gradient at 4°C.to that established at 20°C, the 70 per cent concentration would only need to be changed to 65 per cent and the 85 per cent concentration to 82 per cent (Jepsen and Skottun 1982). These are still m u c h higher than 60 per cent and 75 per cent, but would fit the results in Fig 1. Therefore, while the method described by
117
Sedgwick et al (1986) is sound, it is suggested that the recommended concentrations of Percoll should be changed. As with all laboratory techniques, workers must validate this technique in their own laboratories, and adjust it to their particular requirements. It is suggested lhat, depending on operating temperature, they start with the concentrations recommended here or those of Pycock et al (1987). References JEPSEN, L. V. & SKOTTUN, T. (1982) A rapid one-step method for the isolation of human granulocytes from whole blood. Scandinavian Journal of Clinical Laboratory lnvestigation 42,235-238 PYCOCK, J, F., ALLEN, W. E. & MORRIS, T. H, (1987) Rapid, single-step isolation of equine neutrophils on a discontinuous Pereoll density gradient. Research in Veterinary Science 42, 411-412 SEDGWICK, A. D., MORRIS, T., RUSSELL, B. A. & LEES, P. (1986) Single step purification procedure for the rapid separation of equine leucocytes. Veterinary Research Communications 10, 445 -452
Received April 24, 1990 Accepted September 18, 1990
Isolation of equine peripheral blood mononuclear cells using Percoli S. A. MAY*, R. E. H O O K E t , P. LEES, Royal Veterinary College, Hawkshead House, North Mymms,
Hatfield, Hertfordshire AL9 7TA
The concentration of Pereoll required for isolating equine peripheral blood mononuclear cells has been reinvestigated. A poor cell yield was obtained at the 60 per cent concentration already reported. It is recommended that workers specifically interested in high yields of mononuclear cells, for investigation of lymphocyte and monocyte functions, use a concentration of 65 per cent Percoll. However, workers wishing to isolate pure populations of equine neutrophils might consider a concentration of 70 per cent in the upper layer of Percoll used to retain the mononuclear cells.
blood at 4°C gently layered on top of each. The tubes were centrifuged at 440 g for 30 minutes at 4°C, the white cell layer above the Percoll harvested, and total and differential white cell counts performed. The results are displayed in Fig 1. All the mononuclear cells present in the blood were recovered on 65 per cent and 67.5 per cent Percoll, without any neutrophil contamination, compared with 43 per cent of mononuclear cells recovered on 60 per cent Percoll. Although all mononuclear cells were recovered at concentrations above 67.5 per cent, increasing contamination with neutrophils occurred. Therefore, a concentration of 65 per cent Percoll is recommended for extracting mononuclear cells from equine blood. Fig 1 shows that at 4°C a discontinuous gradient of the type recommended by Sedgwick et al (1986) of 60 per cent and 75 per cent Percoll, results in poor recovery of m o n o nuclear cells above the 60 per cent Percoll (although these are pure) and poor recovery of neutrophils, contaminated by the rest of the mononuclear ceils, above the 75 per cent Percoll. The concentration of the upper layer of Percoll must be in the range of 65 to 67.5 per cent if this contamination of the neutrophil layer is to be avoided. Thus, a 65 per cent/75 per cent gradient would yield relatively pure populations of cells, although only 60 per cent of the neutrophils would be recovered on the bottom layer. Another group of workers has described a method for isolating equine neutrophils at room temperature. This involves a discontinuous Percoll gradient of 70 per cent/ 85 per cent (Pycock et a11987). These workers report a purity
IN order to undertake studies on individual cell populations in vitro, it is first necessary to isolate the cells of interest. In the case of blood cells, it is possible to exploit differences in the density of each type of cell to achieve separation of pure populations on continuous or discontinuous density gradients (Pycock et al 1987). The isolation of equine neutrophils and equine mononuclear cells at 4°C on a discontinuous Percoll (Pharmacia) gradient, using 60 per cent Percoll to retain the mononuclear cells and 75 per cent Percoll to retain the neutrophils, has been described, with the authors reporting 99 per cent purity of the mononuclear cell fraction and 94 per cent purity of the neutrophil fraction (Sedgwick et al 1986). However, problems with this technique, involving low yields of mononuclear cells relative to the total numbers present in the original blood, and contamination of the neutrophil fraction with large numbers of mononuclear cells, led us to reinvestigate the optimal density for recovery of the mononuclear cell fraction. The purpose of this communication is to report the density of Percoll required for retention of the whole of the mononuclear cell fraction without significant contamination by neutrophils. The methodology of Sedgwick et al (1986) was retained, except that Hanks' balanced salt solution (HBSS) replaced m i n i m u m essential medium. 'Stock' Percoll was prepared by mixing nine parts Percoll with one part 10 × concentrated HBSS. This stock was diluted further with HBSS, and the concentration expressed as percentage stock Percoll. Thus 6 ml stock Percoll and 4 ml HBSS were designated 60 per cent. A range of Percoll concentrations from 60 to 75 per cent, at 2.5 per cent intervals, was investigated. A volume of 2-5 ml of each concentration of Percoll was placed in centrifuge tubes at 4°C and 2 ml heparinised whole equine
/
*Present address: Division of Equine Studies, University of Liverpool Veterinary Field Station, Leahurst, Neston, Wirral, L64 7TE tPresent address: Duphar Veterinary Ltd, Solvay House, Hedge End, Southampton, SO3 4QH
o~ 60
Q
/ Q ~ , 65 70 Percoll concentration (%)
,
, 75
FIG 1 : The effect of gradient density on white blood cell recovery. • • Total cells, © 0 Neutrophils, ~ A Mononuclear ceils
116
Percoll isolation o f equine b l o o d cells of 98 per cent and a recovery of 84 to 87 per cent. Although, an 85 per cent concentration of Percoll was not investigated in this study, it will be seen by reference to Fig 1 that this is likely to retain all the neutrophils. In addition, it can be seen that the 70 per cent Percoll retains all the mononuclear cells and, in this experiment, about 8 per cent of the neutrophils. This accords with the levels of purity and recovery reported by Pycock et al (1987). Thus, investigators interested mainly in pure populations of neutrophils should choose a slightly higher concentration of Percoll in the upper layer, such as 70 per cent, whereas others wishing to isolate mononuclear cells should choose the concentration of 65 per cent as outlined above. In discussing the difference between their results and those of Sedgwick et al (1986), Pycock et al (1987) cited the effect of temperature on Percoll density (Jepsen and Skottun 1982). However, the results reported here demonstrate that the original concentrations of Sedgwick et al (1986) do not yield the pure populations of cells intended. In addition, to obtain an equivalent gradient at 4°C.to that established at 20°C, the 70 per cent concentration would only need to be changed to 65 per cent and the 85 per cent concentration to 82 per cent (Jepsen and Skottun 1982). These are still m u c h higher than 60 per cent and 75 per cent, but would fit the results in Fig 1. Therefore, while the method described by
117
Sedgwick et al (1986) is sound, it is suggested that the recommended concentrations of Percoll should be changed. As with all laboratory techniques, workers must validate this technique in their own laboratories, and adjust it to their particular requirements. It is suggested lhat, depending on operating temperature, they start with the concentrations recommended here or those of Pycock et al (1987). References JEPSEN, L. V. & SKOTTUN, T. (1982) A rapid one-step method for the isolation of human granulocytes from whole blood. Scandinavian Journal of Clinical Laboratory lnvestigation 42,235-238 PYCOCK, J, F., ALLEN, W. E. & MORRIS, T. H, (1987) Rapid, single-step isolation of equine neutrophils on a discontinuous Pereoll density gradient. Research in Veterinary Science 42, 411-412 SEDGWICK, A. D., MORRIS, T., RUSSELL, B. A. & LEES, P. (1986) Single step purification procedure for the rapid separation of equine leucocytes. Veterinary Research Communications 10, 445 -452
Received April 24, 1990 Accepted September 18, 1990
