DAVID S. CHI and NICK S. HARRIS **. Departments of Human Biological Chemistry and Genetics and Surgery, and Division of. Transplantation Immunology ...
Journal o f Immunological Methods, 19 ( 1 9 7 8 ) 1 6 9 - - 1 7 2
169
© E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press
A SIMPLE M E T H O D F O R T H E I S O L A T I O N O F M U R I N E P E R I P H E R A L BLOOD LYMPHOCYTES *
D A V I D S. CHI a n d N I C K S. H A R R I S **
Departments o f Human Biological Chemistry and Genetics and Surgery, and Division o f Transplantation Immunology, Shriners Burns Institute, University o f Texas Medical Branch, Galveston, T X 77550, U.S.A. ( R e c e i v e d 24 May 1 9 7 7 , a c c e p t e d 9 A u g u s t 1 9 7 7 )
A simple, rapid a n d r e p r o d u c i b l e s e m i - m i c r o t e c h n i q u e was d e v e l o p e d for t h e isolation o f m u r i n e p e r i p h e r a l b l o o d l y m p h o c y t e s . M u r i n e p e r i p h e r a l b l o o d was c o l l e c t e d f r o m b r a c h i a l vessels a n d c e n t r i f u g e d t h r o u g h a F i c o l l - - H y p a q u e g r a d i e n t in a m i c r o f u g e t u b e y i e l d i n g 1--1.5 × 106 l y m p h o c y t e s p e r m o u s e .
INTRODUCTION
Most of the lymphocytes used for immunological assays in murine models are obtained from the spleen, t h y m u s and lymph nodes, due to the small a m o u n t of peripheral blood available. However, in human systems the primary source of lymphocytes is the peripheral blood. Thus, difficulties in interpretation can arise from trying to compare the results of these two different systems. In order to study l y m p h o c y t e s from the peripheral blood of mice we developed a simple, rapid m e t h o d for the isolation of these cells. MATERIALS AND METHODS
Peripheral blood was obtained from individual 12-week-old A/J female mice (Jackson Laboratory, Bar Harbor, ME). The mouse was anesthetized by ether inhalation. It is important that the mouse not be over-anesthetized, since this often results in the reduction of blood flow in the brachial vessels. The peripheral blood was then collected by severing the brachial vessels at the axillary region (Young and Chambers, 1973), dropping 0.1 ml phenolfree heparin at the surgical site to prevent clot formation. Approximately, 1.0 ml of whole blood was obtained. This bleeding m e t h o d requires less skill * This w o r k was s u p p o r t e d in p a r t b y N I H G r a n t R O 1 C A 1 5 2 7 8 02 a n d a g r a n t f r o m t h e Shriners Burns Institute, Galveston Unit. ** To w h o m r e q u e s t s for r e p r i n t s s h o u l d be a d d r e s s e d at S h r i n e r s B u r n s I n s t i t u t e , 6 1 0 T e x a s Aver.ue, G a l v e s t o n , T X 7 7 5 5 0 , U.S.A.
170 than cardiac puncture, and yields a fairly constant volume of peripheral blood. If sterility of l y m p h o c y t e preparations was required for the subsequent immunological assays, an aseptic technique of obtaining peripheral blood was used. Mice were shaved at the axillary region one day prior to bleeding, in order n o t to excite the mice and thus no t to affect the cell c o u n t and the a m o u n t o f blood obtainable. After the mouse was anesthetized it was attached by its limb to a surgical board (Brookline Surgical Specialities, Westford, MA) and the shaved axillary region was cleaned 3 times with 70% alcohol. The skin in the axillary region was lifted with sterile forceps and excised with sterile scissors to expose the pectoralis major and latissimus dorsi muscles. The opened skin was further separated from the muscle using a sterile Q-tip and held in place by separators in order to form a p o c k e t between skin and musculature. The blood collected from this p o c k e t was sterile. This was confirmed by multiple blood culturing assays. A Ficoll--ttypaque gradient of spec. gr. 1.076 g/ml was prepared according to the m e t h o d of BSyum (1968). Two ml of Ficoll--Hypaque gradient was added to small test tubes (Falcon No. 2058, Oxnard, CA). The whole blood was diluted with 3 parts (by volume) of RPMI 1640 medium (Associated Biomedic Systems, Buffalo, NY). The diluted whole blood (approx. 4 ml) from a single mouse was carefully layered o n t o the Ficoll--Hypaque gradient. Th e tube was centrifuged with a horizontal r o t o r at 400 RCF for 25 min at 4°C. The cells at the interface were removed and transferred into a Beckman microfuge B t ube (Beckman Instruments, Inc., Anaheim CA), using a pasteur pipette drawn out to a narrow end. The tube was centrifuged for 4 min using a Beckman centrifuge B. The pellet of cells was then washed three times at 4°C with RPMI 1640 medium. The resuspension of cells after one minute centrifugation was done using a vortex genie mixer (Scientific Products, McGaw Park, ILL RESULTS AND DISCUSSION Various dilutions of whole blood diluted with RPMI 1640 medium were layered o n t o Ficoll--Hypaque gradient. In addition, the length of time and force o f centrifugation of blood-layered Ficoll--Hypaque gradient were also varied. In some cases, considerable platelet cont am i nat i on and red blood cell c o n t a m i n a t i o n were observed in some preparations. The condition of 1 to 4 dilution of whole blood layered on Fieoll--Hypaque gradient and centrifuged at 400 RCF for 25 min was f ound to give the best isolation of l y m p h o c y t e s . Table 1 presents the relative n u m b e r of white blood cells in mouse peripheral blood before and after Ficoll--Hypaque gradient centrifugation. The absolute recovery from the peripheral blood of 6 mice is presented in table 2. Lymp h o c y t e s comprise 96.5% of the F i c o l l - - H y p a q u e / b l o o d interface with 61% recovery after three washes. The viability of the l y m p h o c y t e preparation as d e ter min ed by tt~cpan blue exclusion was greater than 99%. The functional
171 TABLE 1 Characteristics of murine peripheral blood lymphocytes preparation before and after Ficoll--Hypaque gradient centrifugation. The mean and range represent the results of cell preparation from six individual mice. Differentials were determined by the Wright's staining technique. Cell type
Before Ficoll--Hypaque (%)
After Ficoll--Hypaque (%)
Lymphocytes Monocytes Granulocytes
72.4 5.6 22.0
96.5 1.2 2.3
(59--77) (2-- 8) (18--34)
(94--98) (0-- 3) (2-- 3)
viability o f t h e l y m p h o c y t e p r e p a r a t i o n has b e e n c o n f i r m e d t h r o u g h m i t o g e n s t i m u l a t i o n assays ( m a n u s c r i p t in p r e p a r a t i o n ) as well as Ig-bearing cells and p l a s m a cell assays using an i m m u n o f l u o r e s c e n c e t e c h n i q u e (Chi and Harris, 1977). T h e r e c o v e r y r e p o r t e d h e r e is less t h a n t h a t o f m a n as r e p o r t e d b y G o l d r o s e n et al. ( 1 9 7 7 ) . T h e small v o l u m e s o f m u r i n e p e r i p h e r a l b l o o d involved m a y explain t h e relatively low r e c o v e r y d u e t o a f i x e d a m o u n t o f cell loss. H o w e v e r , the a b s o l u t e n u m b e r o f l y m p h o c y t e s r e c o v e r e d f r o m a single m o u s e averaged b e t w e e n 1 to 1.5 X 106 cells w h i c h is s u f f i c i e n t f o r m o s t i m m u n o l o g i c a l assays (table 2). An a d d i t i o n a l a d v a n t a g e o f this m e t h o d was t h a t t h e w h o l e p r o c e d u r e f o r six individual m o u s e p e r i p h e r a l b l o o d l y m p h o c y t e isolations t o o k less t h a n o n e h o u r . In s u m m a r y , we have d e v e l o p e d a s e m i - m i c r o t e c h n i q u e f o r t h e i s o l a t i o n o f l y m p h o c y t e s f r o m m u r i n e p e r i p h e r a l b l o o d . T h e b l o o d was c o l l e c t e d b y severing t h e brachial vessels at t h e axillary region a n d F i c o l l - - H y p a q u e g r a d i e n t c e n t r i f u g a t i o n was d o n e in m i c r o f u g e tubes. This simple, r a p i d and r e p r o d u c i b l e l y m p h o c y t e isolation t e c h n i q u e yields 1--1.5 X 106 l y m p h o c y t e s f r o m t h e p e r i p h e r a l b l o o d o f an individual m o u s e w h i c h can be used f o r f u r t h e r i m m u n o l o g i c a l assays.
TABLE 2 Recovery of murine peripheral blood lymphocyte preparation after Ficoll--Hypaque gradient centrifugation. The mean and range represent the results of cell preparation from from six individual mice. Cell type
Lymphocytes Monocytes Granulocytes
Cell numbers (× 106)/mouse Before Ficoll--Hypaque
After Ficoll--Hypaque
1.891 (1.148--2.772) 0.136 {0.072--0.222) 0.555 (0.287--0.952)
1.063 (0.524--1.465) 0.012 (0 --0.037) 0.026 (0.011--0.037)
Recovery (%)
60.8 (43.5--97.2) 8.8 ( 0 --20.8) 4.7 ( 3.8-- 7.0)
172 REFERENCES BSyum, A., 1968, J. Clin. Lab. Invest. 21, Suppl. 97, 31. Chi, D.S. and N.S. Harris, 1977, Cancer Res. 33, 1119. Goldrosen, M.H., P.J. Gannon, Mari Lutz and E.D. Holyoke, 1977, J. Immunol. Method~, 14, 15. Young, L. and T.R. Chambers, 1973, Lab. Animal Sci. 23,428.
