Journal of Immunological Methods, 23 (1978)7--15
7
© Elsevier/North-Holland Biomedical Press
A RAPID PHOTOELECTRIC METHOD FOR READING CELL MIGRATION
S. GAUTHIER-RAHMANand J.L. MORLAT Institut d'Immuno-Biologie, Hbpital Broussais, 96 rue Didot, 75674 Paris C~dex 14, Ingdnieur ESO, 125 Boulevard Davout, 75020 Paris, France (Received 27 January 1978, accepted 28 February 1978) A rapid photoelectric method for reading macrophage migration is described which eliminates drawing and planimetry of cell fans. The results obtained are shown to be concordant with those obtained by planimetry. The time required to read a cell migration experiment is reduced from several hours to a few minutes. INTRODUCTION Inhibition of migration of peritoneal exudate cells from a sensitized animal in the presence of the relevant antigen is considered one of the 'in vitro' correlates of delayed hypersensitivity. Since its description by Bloom and Bennett (1966) attempts have been made to standardize and simplify the method. The usual m e t h o d of computing inhibition by projection microscopy, drawing and planimetry is time-consuming. It also imparts an observer bias as the periphery of irregularly shaped cell fans is not easily defined. Rapid methods for reading migration inhibition tests, leukocyte or macrophage, are reported (Jones, 1973; Pekarek and Krejli, 1974; Doble, 1977). A photometric method, remarkable for its rapidity, was described by Williams et al. (1972), but does not seem to have come into wide use. A different photoelectric procedure has been developed in this laboratory. Principle o f the p h o t o e l e c t r i c m e t h o d
Capillary tubes with migrating cells, each m o u n t e d in an individual well of a sterilin migration plate, are viewed under dark-field illumination with a low power (X2.5) microscope (Model N 300, Nachet, Levallois-Perret, France). The image of the cell fan is then directed to the photosensitive surface in the polaroid camera of an apparatus for automatic microphotography (Nachet, Model NS 901). Each cell fan is so oriented that its glass capillary, which diffracts light intensely, is masked by a black rectangle of the same diameter as the capillary affixed to the photographic ocular. Under suitable conditions the migrating cell fan, brightly illuminated in a dark field, is seen within the limits of the rectangular frame which delineates the photosensitive surface and may, if required, be photographed for planimetry.
The light diffracted by the cell fan is measured as electrical potential by the electronic exposure meter of the apparatus (Nachet NS 901) connected to a volt meter of sufficient sensitivity. The voltage observed corresponds to the a m o u n t of light diffracted. A background value is obtained as the.mean of 8--12 wells containing medium alone. The a m o u n t of light (in lumens) diffracted by a cell fan is calculated as follows: Let VT be the voltage observed and ~ the a m o u n t of light, then VT = K log 1/¢ Now ¢ = ~c + CB where ~c is the light diffracted by the cells alone and ~B is the background (light from the microscope, defects in the sterilin plate, small air bubbles, etc.) 1 So that VT = K log ~c + ~B ~c + ~B = 10 V w and~B = 1 0 V B where VB is the background voltage. Hence ~c = 10 vw -- 10vB In general when the migrations are brightly lit ~B is very small compared to ~c. MATERIALS AND METHODS
Animals Hartley guinea pigs, in groups of 3--5 animals weighing about 0.5 kg, were immunized with 0.25 ml of Freund's complete adjuvant mixed with 10 gg ovalbumin in 0.25 ml saline, by intradermal injection (i.d.) on both sides of the shaven back and cells obtained 4--6 weeks later (positive experiments). Hartley guinea pigs treated with C. parvum 500 t~g mixed with ovalbumin (Ov), 10 pg i.d. on day 0 and given 3 booster injections of ovalbumin 10 gg i.d. on days 28, 92 and 233 were sacrificed on day 408 (negative experiment). At this late stage of immunization with C. parvum, delayed hypersensitivity was no longer observed, dlthough an enhanced amnestic response was still fully present.
Preparation of capillaries Peritoneal exudate cells were obtained on day 6 after the intraperitoneal injection of 20 ml of thioglycollate, washed twice in ice-cold Medium 199 containing .10% decomplemented fetal calf serum (FCS) and antibiotics and
2 5 U / m l calciparine, and a third time in the same medium w i t h o u t calciparine. The cell viability and ratio of red to white cells was determined by counting in presence of trypan blue. The final cell suspension, in Medium 199 with 50% FCS w i t h o u t calciparine, was adjusted to about 30--40 X 106 viable cells/ml. The cell suspension, kept in ice, was drawn into hematocrit capillary tubes of internal diameter 1 mm, length 75 m m (Polylabo, Block and Cie, Strasbourg) about 40 pl per capillary, and the ends sealed by flaming. Care was taken to keep the capillary tubes horizontal and not to heat the cell suspension during this procedure. Sealed capillary tubes were laid horizontally in a Petri dish kept on ice. All sealed capillary tubes were centrifuged at the same time at 1500 rpm for 5 min and held in ice thereafter. Eight different concentrations of ovalbumin in Medium 199 with 50% FCS were prepared by adding 0.1 ml of an appropriate concentration of the antigen in Sterile physiological saline to 10 ml of medium, the final concentrations being 0.1 ug, 1 pg, 10 gg, 100 pg, 250 gg/ml. Dry weighed ovalbumin was added to medium to obtain concentrations of 1 mg, 5 mg and 10 mg/ml. All solutions were kept in an ice bath. Each capillary tube was cut below the level of the red blood cells and immediately m o u n t e d in 0.4 ml of medium in a well in a previously prepared sterilin migration plate; 8--12 capillaries were prepared for each concentration of antigen. Control capillaries, 8--16 in number, were m o u n t e d at the beginning and at the end of the experiment. The plates were incubated at 37°C and read at 15--18 h and on day 2. All the capillaries in each experiment were read photoelectrically in less than 45 rain, each fan area was then drawn and measured by planimetry. Percentage inhibition was calculated as follows: % Inhibition:
SC _ Soy X 100 Sc
where SO: mean area of control capillaries; S°V: mean area of capillaries at a given antigen concentration. A similar calculation was done for lumens: L~: mean luminosity of control capillaries; L°V: mean luminosity of capillaries at a given antigen concentration. L c _ LOV % Inhibition: - - X 100. L~ RESULTS
It was found that the two methods gave similar results. When definite inhibition was present it was observed in the same zone of antigen concentration by both methods and the per cent inhibition was similar if not identical. When inhibition was absent by planimetry, the same was true by the photoelectric m e t h o d (Table 1).
10 TABLE 1 C O M P A R I S O N O F T H E E F F E C T O F D I F F E R E N T A N T I G E N C O N C E N T R A T I O N S ON T H E M I G R A T I O N O F M A C R O P H A G E S A S O B S E R V E D BY T H E P L A N I M E T R I C A N D T H E P H O T O E L E C T R I C M E T H O D S IN A R E P R E S E N T A T I V E E X P E R I M E N T Twelve capillaries p e r a n t i g e n c o n c e n t r a t i o n a n d 15 c o n t r o l s . I n h i b i t i o n is seen t o b e a f u n c t i o n o f a n t i g e n c o n c e n t r a t i o n , t h e o p t i m a l c o n c e n t r a t i o n h e r e b e i n g 250 pg/ml. In t h e p r e s e n c e o f very l o w a n t i g e n c o n c e n t r a t i o n s (0.1 pg, 1 pg) s t i m u l a t i o n occurs. Definite i n h i b i t i o n at n o n - o p t i m a l a n t i g e n c o n c e n t r a t i o n s is d e t e c t e d earlier b y t h e p h o t o electric m e t h o d (10 pg, 5 mg, see c o l u m n s 2 a n d 4 ) a n d is practically m a x i m a l at 18 h at the optimal concentration by this method. P e r c e n t effect as o b s e r v e d
Ovalbumin concentration (pg/ml)
By p l a n i m e t r y 18 h
0 (controls) 0,1 1 10 100 250 103 5 × 103
25
.
48 h .
. +6.1 --4.1 --26.5 --40.7 --51.6 --42.8 --38.3
+19.7 +5.3 --13.4 --30.0 --39.4 --20.0 --17.1
•
By l u m i n o s i t y 18 h
48 h
+10.8 --5.4 --19.2 --28.0 --37.3 --23.7 --21.3
+6.6 --3.2 --19.5 --27.4 --39.5 --24.9 --26.5
.
¢--~r
Surface
end
Lumens
25
e~r
20
20
15
15
¢
3
Q
=l
10
10
0D
=1
5 0
~/ ~r Controls
i
-1
I
0
Log1.° ~ c .
i
I
1
2
•
i
3
Ovallmmm
•
I
. 1 0 -3
4
•glml
Fig. 1. T h e m e a n surface area a n d t h e m e a n l u m i n o s i t y of m i g r a t i n g cell fans at d i f f e r e n t a n t i g e n c o n c e n t r a t i o n s w i t h s t a n d a r d d e v i a t i o n s , in a positive e x p e r i m e n t . R e a d i n g s at 18h.
11
Fig. 1 shows the parallelism between surface area and luminosity at different antigen concentrations and Fig. 2 the per cent inhibition curves obtained by the two methods in a representative positive experiment. There were 12 capillaries for each antigen concentration and 15 controls. The per cent inhibition observed at 18 h and 48 h is shown. Maximal inhibition as observed by the photoelectric m e t h o d is already present at 18 h, whereas by the planimetric m e t h o d the inhibition at 48 h is greater than on day 1.
Effect of culture duration Cell fans read at 15 or 18 h were found to be still within the limits of the photosensitive frame and could have been read earlier. At 48 h in some experiments the outer border of some fans was no longer within these limits, although in the experiment presented in Fig. 2 this was not the case. Good concordance was obtained between surface area and lumens within experiments, in spite of some dispersion of individual readings (Fig. 3). Dispersion was greater at 48 h. Between 18 and 48 h the surface area of the migrations increased much more than their luminosity. For control capillaries the surface area more than doubled, whereas luminosity increased by less than 40% (Table 2). This
0,=0 e~-e
Lumens Surface
50
A 18 hours
40
48 hours
•
2
3
6000
30 :5
;
20
2
0
÷!
-1 /J' (?oOte.'
o,t
10
/
10
1
2
3
4
-1,~ 0
-~g
1
4
itII 20
•
Log
Conc.
Ovalbumin
~Jlml
Fig. 2. The per cent inhibition observed at different antigen concentrations by the photoelectric method compared with planimetry, at 18 h and 48 h.
At 18 h
18.96 22.72 19.98 16.41 13.25 11.48 15.16 15.70
15 12 12 12 12 12 12 12
0 (controls) 0, 1 1 10 100 250 103 5 × 103
(_+3.15) (_+2.58) (_+3.75) (-+2.58) (_+1.70) (_+1.76) (-+2.74) (_+3.11)
(cm 2 )
Mean surface
No. o f capillaries
Ovalbumin concentration (~g/ml)
41.11 43.63 39.40 30.28 24.38 19.16 23.57 25.43
At 48 h
(_+10.05) (+3.73) (_+4.48) (_+5.00) (_+2.72) (-+4.06) (_+4.63) (_+3.65)
(cm 2)
116.5 92.5 97.2 84.6 84.4 74.2 55.4 62.0
Per cent increase 20,578 22,810 19,454 16,658 14,804 12,720 15,708 16,204
At 18 h
(_+4527) (_+2456) (-+3712) (-+3630) (+2766) (_+2973) (-+2902) (+3078)
(Lumens)
Mean luminosity
27,162 28,963 26,280 21,875 19,763 16,484 20,413 19,800
At 48 h
(_+6929) (_+5340) (_+4596) (+4557) (-+3150) (_+3411) (_+3549) (_+3399)
(Lumens)
32.5 27.0 36.0 31.4 33.4 29.6 29.9 22.2
Per cent increase
RELATIONSHIP BETWEEN S U R F ACE A R E A AND LUMINOSITY OF CELL FANS AT 18 h AND 48 h IN A R E P R E S E N T A T I V E EXPERIMENT Inhibited cell fans increased less in surface between 18 h and 48 h than non-inhibited fans, whereas the increase in luminosity was about the same. This is shown by the different shapes of the surface area/luminosity plots at 18 h and 48 h and accounts for the greater inhibition at 48 h as observed by planimetry (Fig. 3 and Table 1).
TABLE 2
13 60
o o
L
40
o
I
N"" L
(/'j
S
g ,~/'~°f" o
:i
O,
xl 0 .3
b-
oo
48° hours o
o
18 hours
I
I
10
20
I
30
40
Lumens Fig. 3. T h e surface area o f 100 cell fans p l o t t e d against their l u m i n o s i t y at 18 and 48 h.
•
Surface
0 Lumens
3O
20
20 •
re-
3 {D
10
0
xlO -e
10
'
10 Cell
I
20
I
3O
!
I
50
40
Concentration
x l O -3
/ml
Fig. 4. R e l a t i o n s h i p b e t w e e n surface area or l u m i n o s i t y o f c o n t r o l cell fans and cellular c o n c e n t r a t i o n as o b t a i n e d in d i f f e r e n t e x p e r i m e n t s .
14 means that the migrating cells, as indicated by the light reflected by them, had for the most part already migrated out of the capillary by 18 h and much of the increase in area observed by day 2 was due to their spreading out rather than to the arrival of new cells. This is shown by the different slopes of the surface area/lumen plots at 18 and 48 h (Fig. 3).
Effect of lysis of red cells It was t h o u g h t that the presence of coloured red cells might influence the photoelectric reading. Cell suspensions containing more than 3 red cells to 1 white cell were not used. An experiment was done in which red cells were lysed with cold NH4C1 0.83% at pH 7 for 10 min. The results obtained with this suspension were comparable to those with an unlysed suspension from the same group of animals.
Effect of cell concentration Cell suspensions used were for the most part between 30--42 × 106 cells/ ml. Other conditions being kept constant the light reflected by the cell fans at 18 h was found to be directly proportional to the cell concentration, whereas the surface area of migrations of cell suspensions of similar concentration was found to vary quite widely. Some suspensions of higher concentration migrated poorly (Fig. 4). DISCUSSION In a detailed statistical analysis of cell migration from capillaries, Morley (1974) has shown that 4, 6 and 9 replicates of both inhibited and control capillaries are required to achieve significance for a reduction of migration index to 0.8 (i.e. 20% inhibition) when the standard deviation of the experim e n t is respectively 10, 15 and 20% (95% confidence limits). In m a n y migration tests only 4 or even 3 capillaries are used and the number of different antigen concentrations is limited. In our experiments a wide range of antigen concentrations, 8--12 capillaries per concentration, was used. The standard deviations observed (Tables 1 and 2) were not larger for the photoelectric m e t h o d than for planimetry, and were homogenous for each type of experiment. The per cent inhibitions observed were similar and statistically significant by both methods in the positive experiments. Negative experiments were negative by both methods. The photoelectric m e t h o d allows migrations to be read as early as 15 h, if n o t earlier, and the readings obtained are similar to those of day 2. The plot of luminosity of control capillaries against cell concentration (each capillary being prepared with about 40 td of suspension) is a straight line, whereas the plot o f surface area against cell concentration is not. Thus the mean surface area of migration from control capillaries, which serves as the basis for calculating inhibition, is n o t constant. It is a c o m m o n experience that for ill-defined reasons cells in some experiments do not migrate, or the area is so small that
15
calculations have a large coefficient of variation. The photoelectric method with constant readings for control fans appears to bypass this difficulty. In conclusion, the photoelectric method is not only time-saving but appears to avoid some delays and pitfalls in cell migration experiments. The time saved is appreciable when it is remembered that drawing, followed by planimetry of 100 cell fans, takes 6 h or more, depending on the operator, whereas the same fans can be read photoelectrically in about half an hour. REFERENCES Bloom, B.R. and B. Bennett, 1966, Science 153, 80. Doble, A.T.A., 1977, J. Immunol. Methods 16, 299. Jones, B.M., 1973, Met. Lab. Technol. 30, 245. Morley, J., 1974, Acta Allergol. 29, 185. Pekarek, J. and J. Krejli, 1974, J. Immunol. Methods 6, 1. Williams, T.J., J. Morley and V. Wolstencroft, 1972, J. Immunol. Methods 2, 137.
7
© Elsevier/North-Holland Biomedical Press
A RAPID PHOTOELECTRIC METHOD FOR READING CELL MIGRATION
S. GAUTHIER-RAHMANand J.L. MORLAT Institut d'Immuno-Biologie, Hbpital Broussais, 96 rue Didot, 75674 Paris C~dex 14, Ingdnieur ESO, 125 Boulevard Davout, 75020 Paris, France (Received 27 January 1978, accepted 28 February 1978) A rapid photoelectric method for reading macrophage migration is described which eliminates drawing and planimetry of cell fans. The results obtained are shown to be concordant with those obtained by planimetry. The time required to read a cell migration experiment is reduced from several hours to a few minutes. INTRODUCTION Inhibition of migration of peritoneal exudate cells from a sensitized animal in the presence of the relevant antigen is considered one of the 'in vitro' correlates of delayed hypersensitivity. Since its description by Bloom and Bennett (1966) attempts have been made to standardize and simplify the method. The usual m e t h o d of computing inhibition by projection microscopy, drawing and planimetry is time-consuming. It also imparts an observer bias as the periphery of irregularly shaped cell fans is not easily defined. Rapid methods for reading migration inhibition tests, leukocyte or macrophage, are reported (Jones, 1973; Pekarek and Krejli, 1974; Doble, 1977). A photometric method, remarkable for its rapidity, was described by Williams et al. (1972), but does not seem to have come into wide use. A different photoelectric procedure has been developed in this laboratory. Principle o f the p h o t o e l e c t r i c m e t h o d
Capillary tubes with migrating cells, each m o u n t e d in an individual well of a sterilin migration plate, are viewed under dark-field illumination with a low power (X2.5) microscope (Model N 300, Nachet, Levallois-Perret, France). The image of the cell fan is then directed to the photosensitive surface in the polaroid camera of an apparatus for automatic microphotography (Nachet, Model NS 901). Each cell fan is so oriented that its glass capillary, which diffracts light intensely, is masked by a black rectangle of the same diameter as the capillary affixed to the photographic ocular. Under suitable conditions the migrating cell fan, brightly illuminated in a dark field, is seen within the limits of the rectangular frame which delineates the photosensitive surface and may, if required, be photographed for planimetry.
The light diffracted by the cell fan is measured as electrical potential by the electronic exposure meter of the apparatus (Nachet NS 901) connected to a volt meter of sufficient sensitivity. The voltage observed corresponds to the a m o u n t of light diffracted. A background value is obtained as the.mean of 8--12 wells containing medium alone. The a m o u n t of light (in lumens) diffracted by a cell fan is calculated as follows: Let VT be the voltage observed and ~ the a m o u n t of light, then VT = K log 1/¢ Now ¢ = ~c + CB where ~c is the light diffracted by the cells alone and ~B is the background (light from the microscope, defects in the sterilin plate, small air bubbles, etc.) 1 So that VT = K log ~c + ~B ~c + ~B = 10 V w and~B = 1 0 V B where VB is the background voltage. Hence ~c = 10 vw -- 10vB In general when the migrations are brightly lit ~B is very small compared to ~c. MATERIALS AND METHODS
Animals Hartley guinea pigs, in groups of 3--5 animals weighing about 0.5 kg, were immunized with 0.25 ml of Freund's complete adjuvant mixed with 10 gg ovalbumin in 0.25 ml saline, by intradermal injection (i.d.) on both sides of the shaven back and cells obtained 4--6 weeks later (positive experiments). Hartley guinea pigs treated with C. parvum 500 t~g mixed with ovalbumin (Ov), 10 pg i.d. on day 0 and given 3 booster injections of ovalbumin 10 gg i.d. on days 28, 92 and 233 were sacrificed on day 408 (negative experiment). At this late stage of immunization with C. parvum, delayed hypersensitivity was no longer observed, dlthough an enhanced amnestic response was still fully present.
Preparation of capillaries Peritoneal exudate cells were obtained on day 6 after the intraperitoneal injection of 20 ml of thioglycollate, washed twice in ice-cold Medium 199 containing .10% decomplemented fetal calf serum (FCS) and antibiotics and
2 5 U / m l calciparine, and a third time in the same medium w i t h o u t calciparine. The cell viability and ratio of red to white cells was determined by counting in presence of trypan blue. The final cell suspension, in Medium 199 with 50% FCS w i t h o u t calciparine, was adjusted to about 30--40 X 106 viable cells/ml. The cell suspension, kept in ice, was drawn into hematocrit capillary tubes of internal diameter 1 mm, length 75 m m (Polylabo, Block and Cie, Strasbourg) about 40 pl per capillary, and the ends sealed by flaming. Care was taken to keep the capillary tubes horizontal and not to heat the cell suspension during this procedure. Sealed capillary tubes were laid horizontally in a Petri dish kept on ice. All sealed capillary tubes were centrifuged at the same time at 1500 rpm for 5 min and held in ice thereafter. Eight different concentrations of ovalbumin in Medium 199 with 50% FCS were prepared by adding 0.1 ml of an appropriate concentration of the antigen in Sterile physiological saline to 10 ml of medium, the final concentrations being 0.1 ug, 1 pg, 10 gg, 100 pg, 250 gg/ml. Dry weighed ovalbumin was added to medium to obtain concentrations of 1 mg, 5 mg and 10 mg/ml. All solutions were kept in an ice bath. Each capillary tube was cut below the level of the red blood cells and immediately m o u n t e d in 0.4 ml of medium in a well in a previously prepared sterilin migration plate; 8--12 capillaries were prepared for each concentration of antigen. Control capillaries, 8--16 in number, were m o u n t e d at the beginning and at the end of the experiment. The plates were incubated at 37°C and read at 15--18 h and on day 2. All the capillaries in each experiment were read photoelectrically in less than 45 rain, each fan area was then drawn and measured by planimetry. Percentage inhibition was calculated as follows: % Inhibition:
SC _ Soy X 100 Sc
where SO: mean area of control capillaries; S°V: mean area of capillaries at a given antigen concentration. A similar calculation was done for lumens: L~: mean luminosity of control capillaries; L°V: mean luminosity of capillaries at a given antigen concentration. L c _ LOV % Inhibition: - - X 100. L~ RESULTS
It was found that the two methods gave similar results. When definite inhibition was present it was observed in the same zone of antigen concentration by both methods and the per cent inhibition was similar if not identical. When inhibition was absent by planimetry, the same was true by the photoelectric m e t h o d (Table 1).
10 TABLE 1 C O M P A R I S O N O F T H E E F F E C T O F D I F F E R E N T A N T I G E N C O N C E N T R A T I O N S ON T H E M I G R A T I O N O F M A C R O P H A G E S A S O B S E R V E D BY T H E P L A N I M E T R I C A N D T H E P H O T O E L E C T R I C M E T H O D S IN A R E P R E S E N T A T I V E E X P E R I M E N T Twelve capillaries p e r a n t i g e n c o n c e n t r a t i o n a n d 15 c o n t r o l s . I n h i b i t i o n is seen t o b e a f u n c t i o n o f a n t i g e n c o n c e n t r a t i o n , t h e o p t i m a l c o n c e n t r a t i o n h e r e b e i n g 250 pg/ml. In t h e p r e s e n c e o f very l o w a n t i g e n c o n c e n t r a t i o n s (0.1 pg, 1 pg) s t i m u l a t i o n occurs. Definite i n h i b i t i o n at n o n - o p t i m a l a n t i g e n c o n c e n t r a t i o n s is d e t e c t e d earlier b y t h e p h o t o electric m e t h o d (10 pg, 5 mg, see c o l u m n s 2 a n d 4 ) a n d is practically m a x i m a l at 18 h at the optimal concentration by this method. P e r c e n t effect as o b s e r v e d
Ovalbumin concentration (pg/ml)
By p l a n i m e t r y 18 h
0 (controls) 0,1 1 10 100 250 103 5 × 103
25
.
48 h .
. +6.1 --4.1 --26.5 --40.7 --51.6 --42.8 --38.3
+19.7 +5.3 --13.4 --30.0 --39.4 --20.0 --17.1
•
By l u m i n o s i t y 18 h
48 h
+10.8 --5.4 --19.2 --28.0 --37.3 --23.7 --21.3
+6.6 --3.2 --19.5 --27.4 --39.5 --24.9 --26.5
.
¢--~r
Surface
end
Lumens
25
e~r
20
20
15
15
¢
3
Q
=l
10
10
0D
=1
5 0
~/ ~r Controls
i
-1
I
0
Log1.° ~ c .
i
I
1
2
•
i
3
Ovallmmm
•
I
. 1 0 -3
4
•glml
Fig. 1. T h e m e a n surface area a n d t h e m e a n l u m i n o s i t y of m i g r a t i n g cell fans at d i f f e r e n t a n t i g e n c o n c e n t r a t i o n s w i t h s t a n d a r d d e v i a t i o n s , in a positive e x p e r i m e n t . R e a d i n g s at 18h.
11
Fig. 1 shows the parallelism between surface area and luminosity at different antigen concentrations and Fig. 2 the per cent inhibition curves obtained by the two methods in a representative positive experiment. There were 12 capillaries for each antigen concentration and 15 controls. The per cent inhibition observed at 18 h and 48 h is shown. Maximal inhibition as observed by the photoelectric m e t h o d is already present at 18 h, whereas by the planimetric m e t h o d the inhibition at 48 h is greater than on day 1.
Effect of culture duration Cell fans read at 15 or 18 h were found to be still within the limits of the photosensitive frame and could have been read earlier. At 48 h in some experiments the outer border of some fans was no longer within these limits, although in the experiment presented in Fig. 2 this was not the case. Good concordance was obtained between surface area and lumens within experiments, in spite of some dispersion of individual readings (Fig. 3). Dispersion was greater at 48 h. Between 18 and 48 h the surface area of the migrations increased much more than their luminosity. For control capillaries the surface area more than doubled, whereas luminosity increased by less than 40% (Table 2). This
0,=0 e~-e
Lumens Surface
50
A 18 hours
40
48 hours
•
2
3
6000
30 :5
;
20
2
0
÷!
-1 /J' (?oOte.'
o,t
10
/
10
1
2
3
4
-1,~ 0
-~g
1
4
itII 20
•
Log
Conc.
Ovalbumin
~Jlml
Fig. 2. The per cent inhibition observed at different antigen concentrations by the photoelectric method compared with planimetry, at 18 h and 48 h.
At 18 h
18.96 22.72 19.98 16.41 13.25 11.48 15.16 15.70
15 12 12 12 12 12 12 12
0 (controls) 0, 1 1 10 100 250 103 5 × 103
(_+3.15) (_+2.58) (_+3.75) (-+2.58) (_+1.70) (_+1.76) (-+2.74) (_+3.11)
(cm 2 )
Mean surface
No. o f capillaries
Ovalbumin concentration (~g/ml)
41.11 43.63 39.40 30.28 24.38 19.16 23.57 25.43
At 48 h
(_+10.05) (+3.73) (_+4.48) (_+5.00) (_+2.72) (-+4.06) (_+4.63) (_+3.65)
(cm 2)
116.5 92.5 97.2 84.6 84.4 74.2 55.4 62.0
Per cent increase 20,578 22,810 19,454 16,658 14,804 12,720 15,708 16,204
At 18 h
(_+4527) (_+2456) (-+3712) (-+3630) (+2766) (_+2973) (-+2902) (+3078)
(Lumens)
Mean luminosity
27,162 28,963 26,280 21,875 19,763 16,484 20,413 19,800
At 48 h
(_+6929) (_+5340) (_+4596) (+4557) (-+3150) (_+3411) (_+3549) (_+3399)
(Lumens)
32.5 27.0 36.0 31.4 33.4 29.6 29.9 22.2
Per cent increase
RELATIONSHIP BETWEEN S U R F ACE A R E A AND LUMINOSITY OF CELL FANS AT 18 h AND 48 h IN A R E P R E S E N T A T I V E EXPERIMENT Inhibited cell fans increased less in surface between 18 h and 48 h than non-inhibited fans, whereas the increase in luminosity was about the same. This is shown by the different shapes of the surface area/luminosity plots at 18 h and 48 h and accounts for the greater inhibition at 48 h as observed by planimetry (Fig. 3 and Table 1).
TABLE 2
13 60
o o
L
40
o
I
N"" L
(/'j
S
g ,~/'~°f" o
:i
O,
xl 0 .3
b-
oo
48° hours o
o
18 hours
I
I
10
20
I
30
40
Lumens Fig. 3. T h e surface area o f 100 cell fans p l o t t e d against their l u m i n o s i t y at 18 and 48 h.
•
Surface
0 Lumens
3O
20
20 •
re-
3 {D
10
0
xlO -e
10
'
10 Cell
I
20
I
3O
!
I
50
40
Concentration
x l O -3
/ml
Fig. 4. R e l a t i o n s h i p b e t w e e n surface area or l u m i n o s i t y o f c o n t r o l cell fans and cellular c o n c e n t r a t i o n as o b t a i n e d in d i f f e r e n t e x p e r i m e n t s .
14 means that the migrating cells, as indicated by the light reflected by them, had for the most part already migrated out of the capillary by 18 h and much of the increase in area observed by day 2 was due to their spreading out rather than to the arrival of new cells. This is shown by the different slopes of the surface area/lumen plots at 18 and 48 h (Fig. 3).
Effect of lysis of red cells It was t h o u g h t that the presence of coloured red cells might influence the photoelectric reading. Cell suspensions containing more than 3 red cells to 1 white cell were not used. An experiment was done in which red cells were lysed with cold NH4C1 0.83% at pH 7 for 10 min. The results obtained with this suspension were comparable to those with an unlysed suspension from the same group of animals.
Effect of cell concentration Cell suspensions used were for the most part between 30--42 × 106 cells/ ml. Other conditions being kept constant the light reflected by the cell fans at 18 h was found to be directly proportional to the cell concentration, whereas the surface area of migrations of cell suspensions of similar concentration was found to vary quite widely. Some suspensions of higher concentration migrated poorly (Fig. 4). DISCUSSION In a detailed statistical analysis of cell migration from capillaries, Morley (1974) has shown that 4, 6 and 9 replicates of both inhibited and control capillaries are required to achieve significance for a reduction of migration index to 0.8 (i.e. 20% inhibition) when the standard deviation of the experim e n t is respectively 10, 15 and 20% (95% confidence limits). In m a n y migration tests only 4 or even 3 capillaries are used and the number of different antigen concentrations is limited. In our experiments a wide range of antigen concentrations, 8--12 capillaries per concentration, was used. The standard deviations observed (Tables 1 and 2) were not larger for the photoelectric m e t h o d than for planimetry, and were homogenous for each type of experiment. The per cent inhibitions observed were similar and statistically significant by both methods in the positive experiments. Negative experiments were negative by both methods. The photoelectric m e t h o d allows migrations to be read as early as 15 h, if n o t earlier, and the readings obtained are similar to those of day 2. The plot of luminosity of control capillaries against cell concentration (each capillary being prepared with about 40 td of suspension) is a straight line, whereas the plot o f surface area against cell concentration is not. Thus the mean surface area of migration from control capillaries, which serves as the basis for calculating inhibition, is n o t constant. It is a c o m m o n experience that for ill-defined reasons cells in some experiments do not migrate, or the area is so small that
15
calculations have a large coefficient of variation. The photoelectric method with constant readings for control fans appears to bypass this difficulty. In conclusion, the photoelectric method is not only time-saving but appears to avoid some delays and pitfalls in cell migration experiments. The time saved is appreciable when it is remembered that drawing, followed by planimetry of 100 cell fans, takes 6 h or more, depending on the operator, whereas the same fans can be read photoelectrically in about half an hour. REFERENCES Bloom, B.R. and B. Bennett, 1966, Science 153, 80. Doble, A.T.A., 1977, J. Immunol. Methods 16, 299. Jones, B.M., 1973, Met. Lab. Technol. 30, 245. Morley, J., 1974, Acta Allergol. 29, 185. Pekarek, J. and J. Krejli, 1974, J. Immunol. Methods 6, 1. Williams, T.J., J. Morley and V. Wolstencroft, 1972, J. Immunol. Methods 2, 137.
