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A simple technique for simultaneous human leukocytes separation
Journal of Im munological Me th ods, 40 ( 1981 ) 115--116
115
© Elsevier/North-Holland Biomedical Press
A SIMPLE T E C H N I Q U E F O R SI M U L T A N EO U S HUMAN L E U K O C Y T E S SEPARATION
HELENA M.S. BICALHO, CRISTIANO M. GONTIJO and J.A. NOGUEIRA-MACHADO Departamento de Bioqulmica-Imunologia, Instituto de Cidncias Biol6gicas, Universidade Federal de Minas Gerais, C.P. 2486, 30000 Belo Horizonte, MG, Brazil
(Received I August 1980, accepted 6 August 1980)
By centrifuging heparinised human blood through a discontinuous Ficoll-Hypaque density gradient, it is possible to separate simultaneously mononuclear cells, neutrophils and eosinophils.
We have co mb i ne d and modified the techniques divised by T h o r s b y and Bratlie (1970) and Heslop (1978) for the separation of blood cells, based on the centrifugation of cells over a multiple discontinuous gradient, in order to obtain, simultaneously, t he separation of m o n o n u c l e a r cells, neutrophils and eosinophils. The discontinuous gradient was prepared as described below from the following starting solutions: (A) 15.0 ml of 9.0% Ficoll (Sigma Chemical Company, St. Louis, MO) plus 10.0 ml of 50.0% H y p a q u e (Winthrop Products Inc., New York) (density = 1.14); (B) 17.5 ml o f 9.0% Ficoll p l u s 10.0 ml of 50.0% H y p a q u e (dens. = 1.13); (C) 20.0 ml of 9.0% Ficoll plus 10.0 ml o f 50.0% H y p a q u e (dens. = 1.12); and (D) 24.0 ml of 9.0% Ficoll plus 10.0 ml o f 33.9% H y p a q u e (dens. = 1.08). In a 10 ml siliconized glass centrifuge tube, 2.0 ml of each solution A, B, C and D were added sequentially and finally, 2.0 ml of heparinised blood diluted 1 : 2 with 0.9% sodium chloride. After centrifugation at 1000 × g for 40 min at r o o m t e m p e r a t u r e , 4 different cell fractions were obtained at the interface o f each solution. In this way, the interface between p l a s m a and solution D exhibited the m o n o n u c l e a r cell rich fraction. The interface between solution C and D is a neutrophil rich fraction. The interface B-C consists of a mixt ur e of neutrophils and eosinophils. Finally, an eosinophil rich fraction is obtained between solution A and B. The e r y t h r o c y t e s sedim e n t as a pellet at the b o t t o m of the tube. A differential c o u n t was estimated by using smears stained with Giemsa. For eosinophil counts, Disc o m b e ' s m e t h o d was also used. Viability was p e r f o r m e d by means of the t ry pan blue exclusion test. N o t less than 95% of the cells were alive for each test. The purification of bl ood cells by this t echni que with normal differential
116 TABLE 1 Simultaneous separation of human leukocytes by centrifugation through Ficoll-Hypaque d i s c o n t i n u o u s gradients. M = m o n o n u c l e a r cells; N = n e u t r o p h i l s ; Eo = eosinophils. Blood samples
1 2 3 4 5 6 7 8 9 10
Interfaces P l a s m a / D (%)
D/C (%)
C/B (%)
M
N
Eo
M
N
Eo
M
N
Eo
M
N
Eo
100 100 100 100 100 100 100 99 97 100
0 0 0 0 0 0 0 1 3 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
99 98 99 98 96 98 95 96 95 94
1 2 1 2 4 2 5 4 5 6
0 0 0 0 0 0 0 0 0 0
97 98 89 94 80 92 60 84 50 80
3 2 11 6 20 8 40 16 50 20
0 0 0 0 0 0 0 0 0 0
6 3 10 20 10 16 8 10 1 10
94 97 90 80 90 84 92 90 99 90
counting is s h o w n i n T a b l e 1 . T h e p u r i t y cells, 94--99% for neutrophils and 80--99%
B / A (%)
was 97--100% for eosinophils.
for
mononuclear
ACKNOWLEDGEMENTS We appreciate zinelli. This work
the important was supported
advice and assistance by CNPq (Brazil).
of Dr. Giovanni
Gaz-
REFERENCES Heslop, H.E., 1 9 7 8 , J. I m m u n o l . M e t h o d s 2 2 , 3 8 9 . T h o r s b y , E. a n d A. Bratlie, 1 9 7 0 , in: T e r a s a k i H i s t o c o m p a t i b i l i t y T e s t i n g ( M u n k s g a a r d , C o p e n h a g e n ) p. 655.
115
© Elsevier/North-Holland Biomedical Press
A SIMPLE T E C H N I Q U E F O R SI M U L T A N EO U S HUMAN L E U K O C Y T E S SEPARATION
HELENA M.S. BICALHO, CRISTIANO M. GONTIJO and J.A. NOGUEIRA-MACHADO Departamento de Bioqulmica-Imunologia, Instituto de Cidncias Biol6gicas, Universidade Federal de Minas Gerais, C.P. 2486, 30000 Belo Horizonte, MG, Brazil
(Received I August 1980, accepted 6 August 1980)
By centrifuging heparinised human blood through a discontinuous Ficoll-Hypaque density gradient, it is possible to separate simultaneously mononuclear cells, neutrophils and eosinophils.
We have co mb i ne d and modified the techniques divised by T h o r s b y and Bratlie (1970) and Heslop (1978) for the separation of blood cells, based on the centrifugation of cells over a multiple discontinuous gradient, in order to obtain, simultaneously, t he separation of m o n o n u c l e a r cells, neutrophils and eosinophils. The discontinuous gradient was prepared as described below from the following starting solutions: (A) 15.0 ml of 9.0% Ficoll (Sigma Chemical Company, St. Louis, MO) plus 10.0 ml of 50.0% H y p a q u e (Winthrop Products Inc., New York) (density = 1.14); (B) 17.5 ml o f 9.0% Ficoll p l u s 10.0 ml of 50.0% H y p a q u e (dens. = 1.13); (C) 20.0 ml of 9.0% Ficoll plus 10.0 ml o f 50.0% H y p a q u e (dens. = 1.12); and (D) 24.0 ml of 9.0% Ficoll plus 10.0 ml o f 33.9% H y p a q u e (dens. = 1.08). In a 10 ml siliconized glass centrifuge tube, 2.0 ml of each solution A, B, C and D were added sequentially and finally, 2.0 ml of heparinised blood diluted 1 : 2 with 0.9% sodium chloride. After centrifugation at 1000 × g for 40 min at r o o m t e m p e r a t u r e , 4 different cell fractions were obtained at the interface o f each solution. In this way, the interface between p l a s m a and solution D exhibited the m o n o n u c l e a r cell rich fraction. The interface between solution C and D is a neutrophil rich fraction. The interface B-C consists of a mixt ur e of neutrophils and eosinophils. Finally, an eosinophil rich fraction is obtained between solution A and B. The e r y t h r o c y t e s sedim e n t as a pellet at the b o t t o m of the tube. A differential c o u n t was estimated by using smears stained with Giemsa. For eosinophil counts, Disc o m b e ' s m e t h o d was also used. Viability was p e r f o r m e d by means of the t ry pan blue exclusion test. N o t less than 95% of the cells were alive for each test. The purification of bl ood cells by this t echni que with normal differential
116 TABLE 1 Simultaneous separation of human leukocytes by centrifugation through Ficoll-Hypaque d i s c o n t i n u o u s gradients. M = m o n o n u c l e a r cells; N = n e u t r o p h i l s ; Eo = eosinophils. Blood samples
1 2 3 4 5 6 7 8 9 10
Interfaces P l a s m a / D (%)
D/C (%)
C/B (%)
M
N
Eo
M
N
Eo
M
N
Eo
M
N
Eo
100 100 100 100 100 100 100 99 97 100
0 0 0 0 0 0 0 1 3 0
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
99 98 99 98 96 98 95 96 95 94
1 2 1 2 4 2 5 4 5 6
0 0 0 0 0 0 0 0 0 0
97 98 89 94 80 92 60 84 50 80
3 2 11 6 20 8 40 16 50 20
0 0 0 0 0 0 0 0 0 0
6 3 10 20 10 16 8 10 1 10
94 97 90 80 90 84 92 90 99 90
counting is s h o w n i n T a b l e 1 . T h e p u r i t y cells, 94--99% for neutrophils and 80--99%
B / A (%)
was 97--100% for eosinophils.
for
mononuclear
ACKNOWLEDGEMENTS We appreciate zinelli. This work
the important was supported
advice and assistance by CNPq (Brazil).
of Dr. Giovanni
Gaz-
REFERENCES Heslop, H.E., 1 9 7 8 , J. I m m u n o l . M e t h o d s 2 2 , 3 8 9 . T h o r s b y , E. a n d A. Bratlie, 1 9 7 0 , in: T e r a s a k i H i s t o c o m p a t i b i l i t y T e s t i n g ( M u n k s g a a r d , C o p e n h a g e n ) p. 655.