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A colorimetric assay method for the evaluation of neurotrophic activity in vitro
Neuroscienee Letters, 165 (1994) 203 207 ,<) 1994 Elsevier Science Ireland Ltd. All rights reserved 0304-3940/94/$ 07.00
203
NSL 10098
A colorimetric assay method for the evaluation of neurotrophic activity in vitro Yoshiko Ueda*, Ena Walsh**, Hajime Nakanishi, Keizo Yoshida New Drug Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 2-1-6, Kashima, Yodogawa-ku, Osaka 532, Japan (Received 1 July 1993; Revised version received 5 October 1993; Accepted 5 October 19931
Key words:
Neuronal survival: Bioassay: Fibroblast growth factor; Colorimetric assay; Cortex; Tissue culture
A colorimetric assay was established to detect neurotrophic activity by measuring the lysosomal enzyme, acid phosphatase (AP) activity of cultured neuronal cells. Neurons from the cerebral cortex of 14- or 15-day mouse embryo were cultured in serum-free medium for 3 days in 96-well culture plates. A linear relationship was obtained between the AP activity and the number of viable neurons counted under a microscope. The AP assay was used to evaluate the neurotrophic activity of basic fibroblast growth factor. This assay is shown to be simple, sensitive and convenient to detect neurotrophic activity.
Neuronal cell culture in serum-free medium requires the addition of specific neurotrophic factors. They may also be required in vivo to support neuronal cell survival especially alter ischemic injury such as stroke [1] or in association with certain disease such as Alzheimer's disease [2]. Neurotrophic activity has been detected and quantified using in vitro neuronal cell culture. The detection of neurotrophic activity is usually carried out by counting the number of surviving neurons under a microscope. The disadvantages of counting the neuronal cell numbers are that it is time consuming, tedious and not appropriate for the early stage of drug evaluation. Colorimetric assays are widely used to detect cells conveniently. However, only the MTT assay [3] has been reported as a colorimetric assay to determine neuronal cell numbers. Since the number of surviving neurons cultured in serum-free medium decreases very rapidly with time [4], we need a sensitive assay to quantify a small number of neurons in order to detect neurotrophic activity. Recently Connoly reported a colorimetric assay for cellular lysosomal acid phosphatase (AP) with a small number of endothelial cells [5]. They also reported that the optical density (OD) of acid phosphatase activity in a 96-well microplate is directly proportional to the number of viable cells. We modified this assay to evaluate neu-
*Corresponding author.
**Presenl addresx: Elan Corporation plc Monkland Athlone Co., Westmeth, Repubic of Ireland.
ronal cell survival and could detect a slight change in neuronal cell survival with very high sensitivity. Basic fibroblast growth factor (bFGF) has been reported to enhance the survival of primary cultured neurons [6, 7]. Here we detected neurotrophic activity of bFGF by using the colorimetric assay, i.e. acid phosphatase assay in comparison with the microscopic counting assay. Primary culture of cerebral cortical neurons was carried out as described by Walsh et al. [8]. Briefly, the cortex obtained from 14- to 15-day-old fetus of STD:ddy strain mouse (Japan SLC) was dissected free of meninges under sterile conditions and placed in PBS. Cerebral cortices were minced with scissors after rinsing in PBS and were treated with 0.1% trypsin at 37°C for 5 min. Soybean trypsin inhibitor (Sigma) was used to terminate the trypsinization and tissues were triturated with a fire-polished pipette. Cells were centrifuged (300 x g, 4°C for 5 min) and resuspended in DMEM:Ham's F12 (1:1) (Sigma) medium containing 1.2 mg/ml sodium bicarbonate (Ishizu Pharmaceutical Co.), 50 U/ml penicillin, 50 /,zg/ml streptomycin, 2 mM glutamine (Flow laboratories), 100/ag/ml human transferrin (Sigma), 5 yg/ml bovine insulin (Sigma), 20 nM progesterone (Sigma), 30 nM sodium selenite (Sigma) and 100 y M putrescine (Sigma). The viability of cells was monitored by Trypan blue exclusion and was always greater than 85%. The desired numbers of neuronal cells were seeded in 96-well culture plates (Costar 6.4 mm diameter) which had been pre-coated with 5 /ag/ml polylysine (Sigma) overnight. Cells were incubated at 37°C in humidified 5% CO,-95%
204
air for 3 days. Basic fibroblast growth factor (bFGF) from bovine pituitary (Toyobo) was added at initial time of culture and the medium was not exchanged during culture days. Primary cultured neurons had morphological characteristics specific to neurons and an immunohistochemical investigation was carried out using antibody to glial fibrillary acidic protein (GFAP), a specific maker of astrocytes. Immunostaining was done by the method of Ohkuma and colleagues [9] with the following modification. StrAviGen B-SA, an immunostaining kit (BioGenex, USA) which comprises biotinylated second antibody solution and a strepavidin conjugated to peroxidase solution, was used. Cultured neurons seeded in 96-well plates at densities between 1 x 104 and 1 x 105 cells/well were used for determination of cell numbers by counting under a microscope and used for AP assay and M T T assay. Neuronal cell numbers were counted under a microscope using a hemocytometer following detachment with trypsin. To determine the neuronal cell numbers seeded in 96-well plates, 0.1 ml of trypsin solution (0.25% Osaka University) was added to each well to detach the cells. In case of low cell numbers, detached cells from 3 to 12 wells were pooled together and centrifuged at 1500 x g for 10 min and resuspend before counting under a microscope. Neuronal cells cultured in 96-well plates under the same culture conditions for counting assay were used for AP assay. The medium was removed gently by a multichannel pipettor and each well was washed once with 100 /,tl of PBS. Then 100 ,ul of buffer containing 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100, and l0 mM p-nitrophenyl phosphate (Nakarai, Japan) was added to
TABLE 1 THE COMPARISON OF OPTICAL DENSITIES BETWEEN ACID PHOSPHATASE AND MTT ACTIVITY 5 x 104 and I x 105 neurons were seeded in 96-well plates and cultured for ! day to be used either for A P assay or M T T assay. The medium was removed and p-nitrophenyl phosphate was added. The plates were incubated for 3 h at 37°C and the reaction was stopped with 1 N N a O H . The color development was determined at 405 n m (n = 3). For M T T assay, M T T solution was added to the culture medium and then incubated for 4 h at 37°C. The product of M T T was solubitized by acid-isopropanol and the color development was measured at 580 n m (n = 3). Seeded cell no. (cells/well)
Acid phosphatase MTT
OD 5 × 104
I × l0 t
1.154 + 0.018 0.064 + 0.001
2.021 _+ 0.007 0.159 + 0.003
2
r~
,ll
gl, o m
0 0
i
!
i
i
5
I0
15
20
counted cell No. ( x l 0 3) Fig. 1. Relation between AP activity and counted cell number. Neuronal cells were seeded at different densities from 104 to l05 in 96-well plates. After 3-day culture, p-nitrophenyl phosphate was added and incubated at 37°C for 3 h to estimate the A P activity: Neuronal n u m bers were determined by a microscope. Each point shows the mean and S.E.M. for triplicate determinations. The straight line plotted is best fit line calculated.
each well using a multichannel pippetor. The plates were placed in a 37°C 5% CO2-95% air incubator for 3 h. The reaction was stopped by the addition of 10 pl of 1 N NaOH. The color development was determined at 405 nm using a rapid microplate reader (Titertek). M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was described by Mosmann [10] as an automated colorimetric microassay for neurotrophic factors. 5 pl of M T T solution (1.5 mg/ml in culture medium) was added to the culture medium which was converted to an insoluble blue formazan product by living neuronal cells during 4 h incubation. The blue product of M T T is then solubilized by acid-isopropanol (100 pl of 0.04 N HCI in isopropanol) and OD was measured at 580 nm using a microplate reader. Cortical neurons from 14--15 embryonic day were inoculated into 96-well culture plates at different cell numbers. After 1 day culture, these neuronal cells were used for the AP and M T T assays. Connolly reported that the
205
1.0
©
0.8 0.6
©
0.4 0.2
,< 0.0
0
0.3 1 F G F conc.(ng/ml)
0
0.3 1 F G F cone.(ng/ml)
5 4 "-"
3
"~
2
~a
1 0
Fig. 2. The effects of b F G F on survival of primary cultured neurons from the cerebral cortex. 5 x 104 neurons were seeded in 96-well plates and cultured for 3 days with 0, 0.3 and 1 ng/ml bFGF. The number of neurons surviving alter 3 days in culture were measured (a) by AP assay and (b) by direct microscopic counting. Error bars represent S.E.M. for triplicate determinations.
O D of AP activity of endothelial cells increased linearly and was stable for 4 h incubation time [5]. In our study, the AP activity of neurons could be measured linearly up to 3 h incubation time (data not shown). In Table I, we compared the O D observed in the AP assay and the M T T assay measured with 1 day cultured neurons. The O D of M T T assay was very low as compared with AP assay. AP assay was much more sensitive than M T T assay. Next we determined the linearity of the AP assay to quantify the number of neurons. Neurons were seeded at different densities into 96-well plates. After 3 days, a linear relationship between cell numbers and AP activity were observed (Fig. 1). The optical density of the AP assay was linear up to 15000 cells per well.
The neurotrophic activity of b F G F was examined with the AP assay and by counting assay after 3 days in culture. The results are shown in Table II and Fig. 2. The neurotrophic activity of b F G F was dose dependent in both the AP assay and counting assay. The AP activity per 103 neurons remained constant with or without bFGF. Fig. 3 shows the morphological features o1" neurons in the presence or absence of bFGF. Cerebral cortical neurons maintained for 3 days in the presence of 1 ng/ml b F G F exhibited improved survival and stimulated neurite outgrowth in comparison to the control culture. A colorimetric microassay method, measuring AP activity to detect a small neuronal cell numbers to evaluate the neurotrophic activity, is described here. The AP assay previously was used to count low numbers of endothelial cells [5], so we applied this method to determine the neuronal cell numbers in serum-free culture medium. The advantage of this assay is the improved sensitivity to detect a slight change in cultured cell numbers. As shown in Table I, the O D of M T T assay was 0.064, that of AP assay was 1.154, when 5 × 104 neuronal cells were seeded and cultured for 1 day. So the AP assay was approximately 10 times more sensitive than the M T T assay at detecting the changes in neuronal cell numbers. As shown in Fig. 1, AP activity and counted cell number correlated well ( r = 0.996) up to approximately 15000 cells/well. So AP assay was clarified to be relewmt to detect neuronal cell numbers without counting the numbers under a microscope. In this report b F G F was then used as a reference neurotrophic agent. Basic F G F has been reported to enhance the cell survival of hippocampal [11], granule [12], and cerebral cortical neurons [7]. Furthermore, Walicke observed that b F G F also increased the proliferation of astrocytes in culture [6]. In our cultures, contamination
(a)
(b)
Fig, 3. Phase-contrast micrographs of primary cultured cortical neurons. 5 x 10~ neurons were seeded in 96-well plates and cultured for 3 days (a) control and (b) with 1 ng/ml of bFGF. Bar = 100ktm.
206
TABLE I1 THE EFFECT OF bFGF ON CULTURED NEURONS 5 × 104 neurons were seeded in 96-well plates and cultured for 3 days with 0, 0.3 and 1 ng/ml bFGF. The number of neurons surviving after 3 days were measured by AP assay and by direct microscopic counting. Numbers in parentheses were the percentages of control values both in AP assay and counting assay. Data are means and S.E.M. for triplicate determinations. *P < 0.05; **P < 0.01 vs. control (ANOVA). bFGF Conc. (ng/ml)
Acid phosphatase OD
Counted method ( × 10 3 cells/well)
Acid phosphatase OD per 103 cells
0 0.3 1.0
0.608 + 0.003 (100%) 0.764 _+0.030 (129%)** 0.856 + 0.048 (141%)**
3.13 + 0.09 (100%) 3.93 + 0.23 (126%) 4.23 + 0.39 (135%)*
0.19 0.19 0.20
b y a s t r o c y t e s was c h e c k e d for b o t h b y m o r p h o l o g i c a l f e a t u r e u n d e r a m i c r o s c o p e a n d b y using a n t i - G F A P , a m a k e r o f astrocytes. A f t e r 3 d a y s in the absence o r presence o f 1 ng/ml b F G F , few a s t r o c y t e s were o b s e r v e d (Fig. 3). I n a d d i t i o n , less t h a n 3% o f the t o t a l cell p o p u l a t i o n was positive for G F A P ( d a t a n o t shown). M o r r i s o n et al. [7] r e p o r t e d the rate o f c o n t a m i n a t i o n o f a s t r o c y t e s was f r o m 3% to 7% o f t o t a l p o p u l a t i o n after 5 d a y s in culture with or w i t h o u t 1 ng/ml b F G F . O u r results t h a t the c o n t a m i n a t i o n rate with a s t r o c y t e s was less t h a n 3%, was in g o o d a g r e e m e n t with their o b s e r v a t i o n t a k i n g into a c c o u n t the 3 - d a y c u l t u r e p e r i o d in o u r e x p e r i m e n t s . F u r t h e r m o r e the A P activity o f a s t r o c y t e s f r o m m o u s e b r a i n in vivo have been r e p o r t e d to be very low in c o m p a r i s o n with t h a t o f n e u r o n s in vivo [13]. So we s u p p o s e d t h a t the A P activity o f c o n t a m i n a t i n g a s t r o c y t e s was unlikely to have a n y significant influence on o u r results. T h e n e u r o t r o p h i c effect o f b F G F on o u r c u l t u r e d neur o n s was d e t e c t e d b o t h b y A P a s s a y a n d by c o u n t i n g n e u r o n a l cell n u m b e r s . T h e effective c o n c e n t r a t i o n o f b F G F in o u r e x p e r i m e n t s was in g o o d a g r e e m e n t with those r e p o r t e d b y S a i t o [4] a n d W a l i c k [6]. In A P assay, 0.3 ng/ml a n d 1 ng/ml b F G F e n h a n c e d n e u r o n a l survival b y 129% a n d 141% o f c o n t r o l (Table II). In c o u n t i n g assay, e n h a n c e d survival rates were 126% a n d 135% o f c o n t r o l for 0.3 ng/ml a n d 1 ng/ml b F G F , respectively. We s h o w e d t h a t A P activity p e r 103 cells was a l m o s t c o n s t a n t with o r w i t h o u t b F G F w h e n c o m p a r e d in the s a m e e x p e r i m e n t (Table II). W e c a n c o n c l u d e t h a t the n e u r o t r o p h i c effect o f b F G F d e t e c t e d b y A P a s s a y is p r o b a b l y d u e to the i m p r o v e m e n t o f n e u r o n a l survival a n d n o t to the a u g m e n t a t i o n o f A P activity p e r neurons. A P levels h a v e b e e n s h o w n to v a r y u n d e r certain c o n d i tions in o t h e r cell t y p e s a n d culture c o n d i t i o n s . F o r example, A P activity is a m a t u r a t i o n m a r k e r o f m a c r o p h a g e a n d is different a c c o r d i n g to culture ages [14]. So c o n t r o l s s h o u l d be i n c l u d e d in each e x p e r i m e n t to ensure t h a t the e n z y m e levels reflect the a c t u a l cell n u m b e r .
In s u m m a r y we described in this r e p o r t t h a t A P assay is very convenient, r a p i d a n d sensitive assay for detecting n e u r o t r o p h i c factors such as b F G F . 1 Yamada, K., Kinoshita, A., Kohmura, E., Sakaguchi, T., Taguchi, J., Kataoka, K. and Hayakawa, T., Basic fibroblast growth factor prevents thalamic degeneration after cortical infarction, J. Cereb. Blood Flow Metab., 11 (1991)472~,78. 2 Stopa, E.G., Gonzalez, A., Chorsky, R., Corona, R.J., Alvarez. J., Bird, E.D. and Baird, A., Basic fibroblast growth factor in Alzheimer's disease, Biochem. Biophys. Res. Commun., 171 (1990) 690-696. 3 Manthorpe, M., Fagnani, R., Skaper, S.D. and Varon, S., An automated colorimetric microassay for neurotrophic factors, Dev. Brain Res., 25 (1986) 191-198. 4 Abe, K., Takayanagi, M. and Saito, H., Effects of recombinant human basic fibroblast growth factor and its modified protein CS23 on survival of primary cultured neurons from various regions of fetal rat brain, Jpn. J. Pharmacol., 53 (1990) 22b-227. 5 Connolly, D.T., Knight, M.B, Harakas, N.K.. Wittwer, A.J. and Feder, J., Determination of the number of endothelial cells in culture using an acid phosphatase assay, Anal. Biochem., 152 (1986) 136-140. 6 Walicke, P.A. and Baird, A., Neurotrophic effects of basic and acidic fibroblast growth factors are not mediated through glial cells, Dev. Brain Res., 40 (1988) 7179. 7 Morrison, R.S., Sharma, A.. Vellis. J. and Bradshaw. R.A,. Basic fibroblast growth factor supports the survival of cerebral cortical neurons in primary culture, Proc. Natl. Acad. Sci. USA. 83 (1986) 7537--7541. 8 Walsh, E., Ueda, Y., Nakanishi. H. and Yoshida. K.. Neuronal survival and neurite extention supported by astrocytes co-cultured in transwells, Neurosci. Lett.. 138 (1992~ 103-106. 9 Ohkuma, S., Tomono, S., Tanaka, Y.. Kuriyama. K. and Mukainaka, T., Development of taurine biosynthesizing system in cerebral cortical neurons in primary culture, ira. J. Dev. Neurosci.. 4 (1986) 383--395. 10 Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. lmmunol. Methods, 65 (1983) 55-63. 11 Walicke, P., Cowan, W.M., Uneno. N.. Baird. A. and Guillemin. R., FGF promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc. Natl. Acad. Sci. USA. 83 (1986) 3012 3016.
207 12 Hatten, M.E., Lynch, M., Rydel, R.E., Sanchez, J,, Joseph-Silverstein, J., Moscatelli, D. and Rifkin, D.B., In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor, Dev. Biol., 125 (1988) 280-289. 13 Cataldo, A.M., Paskevich, P.A., Kominami, E. and Nixon, R.A., Lysosomal hydrolase of different classes are abnormally distributed
in brains of patient with Alzheimer disease, Proc. Natl. Acad. Sci. USA, 88(1991) 10998 11002. 14 Becker, S., Warren, M.K. and Haskill, S., Colony-stimulation |'actor-induced monocyte survival and differentiation into macrophages in serun free culture. J. Immunol., 139 (1987) 3703 3709.
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NSL 10098
A colorimetric assay method for the evaluation of neurotrophic activity in vitro Yoshiko Ueda*, Ena Walsh**, Hajime Nakanishi, Keizo Yoshida New Drug Research Laboratories, Fujisawa Pharmaceutical Co., Ltd. 2-1-6, Kashima, Yodogawa-ku, Osaka 532, Japan (Received 1 July 1993; Revised version received 5 October 1993; Accepted 5 October 19931
Key words:
Neuronal survival: Bioassay: Fibroblast growth factor; Colorimetric assay; Cortex; Tissue culture
A colorimetric assay was established to detect neurotrophic activity by measuring the lysosomal enzyme, acid phosphatase (AP) activity of cultured neuronal cells. Neurons from the cerebral cortex of 14- or 15-day mouse embryo were cultured in serum-free medium for 3 days in 96-well culture plates. A linear relationship was obtained between the AP activity and the number of viable neurons counted under a microscope. The AP assay was used to evaluate the neurotrophic activity of basic fibroblast growth factor. This assay is shown to be simple, sensitive and convenient to detect neurotrophic activity.
Neuronal cell culture in serum-free medium requires the addition of specific neurotrophic factors. They may also be required in vivo to support neuronal cell survival especially alter ischemic injury such as stroke [1] or in association with certain disease such as Alzheimer's disease [2]. Neurotrophic activity has been detected and quantified using in vitro neuronal cell culture. The detection of neurotrophic activity is usually carried out by counting the number of surviving neurons under a microscope. The disadvantages of counting the neuronal cell numbers are that it is time consuming, tedious and not appropriate for the early stage of drug evaluation. Colorimetric assays are widely used to detect cells conveniently. However, only the MTT assay [3] has been reported as a colorimetric assay to determine neuronal cell numbers. Since the number of surviving neurons cultured in serum-free medium decreases very rapidly with time [4], we need a sensitive assay to quantify a small number of neurons in order to detect neurotrophic activity. Recently Connoly reported a colorimetric assay for cellular lysosomal acid phosphatase (AP) with a small number of endothelial cells [5]. They also reported that the optical density (OD) of acid phosphatase activity in a 96-well microplate is directly proportional to the number of viable cells. We modified this assay to evaluate neu-
*Corresponding author.
**Presenl addresx: Elan Corporation plc Monkland Athlone Co., Westmeth, Repubic of Ireland.
ronal cell survival and could detect a slight change in neuronal cell survival with very high sensitivity. Basic fibroblast growth factor (bFGF) has been reported to enhance the survival of primary cultured neurons [6, 7]. Here we detected neurotrophic activity of bFGF by using the colorimetric assay, i.e. acid phosphatase assay in comparison with the microscopic counting assay. Primary culture of cerebral cortical neurons was carried out as described by Walsh et al. [8]. Briefly, the cortex obtained from 14- to 15-day-old fetus of STD:ddy strain mouse (Japan SLC) was dissected free of meninges under sterile conditions and placed in PBS. Cerebral cortices were minced with scissors after rinsing in PBS and were treated with 0.1% trypsin at 37°C for 5 min. Soybean trypsin inhibitor (Sigma) was used to terminate the trypsinization and tissues were triturated with a fire-polished pipette. Cells were centrifuged (300 x g, 4°C for 5 min) and resuspended in DMEM:Ham's F12 (1:1) (Sigma) medium containing 1.2 mg/ml sodium bicarbonate (Ishizu Pharmaceutical Co.), 50 U/ml penicillin, 50 /,zg/ml streptomycin, 2 mM glutamine (Flow laboratories), 100/ag/ml human transferrin (Sigma), 5 yg/ml bovine insulin (Sigma), 20 nM progesterone (Sigma), 30 nM sodium selenite (Sigma) and 100 y M putrescine (Sigma). The viability of cells was monitored by Trypan blue exclusion and was always greater than 85%. The desired numbers of neuronal cells were seeded in 96-well culture plates (Costar 6.4 mm diameter) which had been pre-coated with 5 /ag/ml polylysine (Sigma) overnight. Cells were incubated at 37°C in humidified 5% CO,-95%
204
air for 3 days. Basic fibroblast growth factor (bFGF) from bovine pituitary (Toyobo) was added at initial time of culture and the medium was not exchanged during culture days. Primary cultured neurons had morphological characteristics specific to neurons and an immunohistochemical investigation was carried out using antibody to glial fibrillary acidic protein (GFAP), a specific maker of astrocytes. Immunostaining was done by the method of Ohkuma and colleagues [9] with the following modification. StrAviGen B-SA, an immunostaining kit (BioGenex, USA) which comprises biotinylated second antibody solution and a strepavidin conjugated to peroxidase solution, was used. Cultured neurons seeded in 96-well plates at densities between 1 x 104 and 1 x 105 cells/well were used for determination of cell numbers by counting under a microscope and used for AP assay and M T T assay. Neuronal cell numbers were counted under a microscope using a hemocytometer following detachment with trypsin. To determine the neuronal cell numbers seeded in 96-well plates, 0.1 ml of trypsin solution (0.25% Osaka University) was added to each well to detach the cells. In case of low cell numbers, detached cells from 3 to 12 wells were pooled together and centrifuged at 1500 x g for 10 min and resuspend before counting under a microscope. Neuronal cells cultured in 96-well plates under the same culture conditions for counting assay were used for AP assay. The medium was removed gently by a multichannel pipettor and each well was washed once with 100 /,tl of PBS. Then 100 ,ul of buffer containing 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100, and l0 mM p-nitrophenyl phosphate (Nakarai, Japan) was added to
TABLE 1 THE COMPARISON OF OPTICAL DENSITIES BETWEEN ACID PHOSPHATASE AND MTT ACTIVITY 5 x 104 and I x 105 neurons were seeded in 96-well plates and cultured for ! day to be used either for A P assay or M T T assay. The medium was removed and p-nitrophenyl phosphate was added. The plates were incubated for 3 h at 37°C and the reaction was stopped with 1 N N a O H . The color development was determined at 405 n m (n = 3). For M T T assay, M T T solution was added to the culture medium and then incubated for 4 h at 37°C. The product of M T T was solubitized by acid-isopropanol and the color development was measured at 580 n m (n = 3). Seeded cell no. (cells/well)
Acid phosphatase MTT
OD 5 × 104
I × l0 t
1.154 + 0.018 0.064 + 0.001
2.021 _+ 0.007 0.159 + 0.003
2
r~
,ll
gl, o m
0 0
i
!
i
i
5
I0
15
20
counted cell No. ( x l 0 3) Fig. 1. Relation between AP activity and counted cell number. Neuronal cells were seeded at different densities from 104 to l05 in 96-well plates. After 3-day culture, p-nitrophenyl phosphate was added and incubated at 37°C for 3 h to estimate the A P activity: Neuronal n u m bers were determined by a microscope. Each point shows the mean and S.E.M. for triplicate determinations. The straight line plotted is best fit line calculated.
each well using a multichannel pippetor. The plates were placed in a 37°C 5% CO2-95% air incubator for 3 h. The reaction was stopped by the addition of 10 pl of 1 N NaOH. The color development was determined at 405 nm using a rapid microplate reader (Titertek). M T T (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was described by Mosmann [10] as an automated colorimetric microassay for neurotrophic factors. 5 pl of M T T solution (1.5 mg/ml in culture medium) was added to the culture medium which was converted to an insoluble blue formazan product by living neuronal cells during 4 h incubation. The blue product of M T T is then solubilized by acid-isopropanol (100 pl of 0.04 N HCI in isopropanol) and OD was measured at 580 nm using a microplate reader. Cortical neurons from 14--15 embryonic day were inoculated into 96-well culture plates at different cell numbers. After 1 day culture, these neuronal cells were used for the AP and M T T assays. Connolly reported that the
205
1.0
©
0.8 0.6
©
0.4 0.2
,< 0.0
0
0.3 1 F G F conc.(ng/ml)
0
0.3 1 F G F cone.(ng/ml)
5 4 "-"
3
"~
2
~a
1 0
Fig. 2. The effects of b F G F on survival of primary cultured neurons from the cerebral cortex. 5 x 104 neurons were seeded in 96-well plates and cultured for 3 days with 0, 0.3 and 1 ng/ml bFGF. The number of neurons surviving alter 3 days in culture were measured (a) by AP assay and (b) by direct microscopic counting. Error bars represent S.E.M. for triplicate determinations.
O D of AP activity of endothelial cells increased linearly and was stable for 4 h incubation time [5]. In our study, the AP activity of neurons could be measured linearly up to 3 h incubation time (data not shown). In Table I, we compared the O D observed in the AP assay and the M T T assay measured with 1 day cultured neurons. The O D of M T T assay was very low as compared with AP assay. AP assay was much more sensitive than M T T assay. Next we determined the linearity of the AP assay to quantify the number of neurons. Neurons were seeded at different densities into 96-well plates. After 3 days, a linear relationship between cell numbers and AP activity were observed (Fig. 1). The optical density of the AP assay was linear up to 15000 cells per well.
The neurotrophic activity of b F G F was examined with the AP assay and by counting assay after 3 days in culture. The results are shown in Table II and Fig. 2. The neurotrophic activity of b F G F was dose dependent in both the AP assay and counting assay. The AP activity per 103 neurons remained constant with or without bFGF. Fig. 3 shows the morphological features o1" neurons in the presence or absence of bFGF. Cerebral cortical neurons maintained for 3 days in the presence of 1 ng/ml b F G F exhibited improved survival and stimulated neurite outgrowth in comparison to the control culture. A colorimetric microassay method, measuring AP activity to detect a small neuronal cell numbers to evaluate the neurotrophic activity, is described here. The AP assay previously was used to count low numbers of endothelial cells [5], so we applied this method to determine the neuronal cell numbers in serum-free culture medium. The advantage of this assay is the improved sensitivity to detect a slight change in cultured cell numbers. As shown in Table I, the O D of M T T assay was 0.064, that of AP assay was 1.154, when 5 × 104 neuronal cells were seeded and cultured for 1 day. So the AP assay was approximately 10 times more sensitive than the M T T assay at detecting the changes in neuronal cell numbers. As shown in Fig. 1, AP activity and counted cell number correlated well ( r = 0.996) up to approximately 15000 cells/well. So AP assay was clarified to be relewmt to detect neuronal cell numbers without counting the numbers under a microscope. In this report b F G F was then used as a reference neurotrophic agent. Basic F G F has been reported to enhance the cell survival of hippocampal [11], granule [12], and cerebral cortical neurons [7]. Furthermore, Walicke observed that b F G F also increased the proliferation of astrocytes in culture [6]. In our cultures, contamination
(a)
(b)
Fig, 3. Phase-contrast micrographs of primary cultured cortical neurons. 5 x 10~ neurons were seeded in 96-well plates and cultured for 3 days (a) control and (b) with 1 ng/ml of bFGF. Bar = 100ktm.
206
TABLE I1 THE EFFECT OF bFGF ON CULTURED NEURONS 5 × 104 neurons were seeded in 96-well plates and cultured for 3 days with 0, 0.3 and 1 ng/ml bFGF. The number of neurons surviving after 3 days were measured by AP assay and by direct microscopic counting. Numbers in parentheses were the percentages of control values both in AP assay and counting assay. Data are means and S.E.M. for triplicate determinations. *P < 0.05; **P < 0.01 vs. control (ANOVA). bFGF Conc. (ng/ml)
Acid phosphatase OD
Counted method ( × 10 3 cells/well)
Acid phosphatase OD per 103 cells
0 0.3 1.0
0.608 + 0.003 (100%) 0.764 _+0.030 (129%)** 0.856 + 0.048 (141%)**
3.13 + 0.09 (100%) 3.93 + 0.23 (126%) 4.23 + 0.39 (135%)*
0.19 0.19 0.20
b y a s t r o c y t e s was c h e c k e d for b o t h b y m o r p h o l o g i c a l f e a t u r e u n d e r a m i c r o s c o p e a n d b y using a n t i - G F A P , a m a k e r o f astrocytes. A f t e r 3 d a y s in the absence o r presence o f 1 ng/ml b F G F , few a s t r o c y t e s were o b s e r v e d (Fig. 3). I n a d d i t i o n , less t h a n 3% o f the t o t a l cell p o p u l a t i o n was positive for G F A P ( d a t a n o t shown). M o r r i s o n et al. [7] r e p o r t e d the rate o f c o n t a m i n a t i o n o f a s t r o c y t e s was f r o m 3% to 7% o f t o t a l p o p u l a t i o n after 5 d a y s in culture with or w i t h o u t 1 ng/ml b F G F . O u r results t h a t the c o n t a m i n a t i o n rate with a s t r o c y t e s was less t h a n 3%, was in g o o d a g r e e m e n t with their o b s e r v a t i o n t a k i n g into a c c o u n t the 3 - d a y c u l t u r e p e r i o d in o u r e x p e r i m e n t s . F u r t h e r m o r e the A P activity o f a s t r o c y t e s f r o m m o u s e b r a i n in vivo have been r e p o r t e d to be very low in c o m p a r i s o n with t h a t o f n e u r o n s in vivo [13]. So we s u p p o s e d t h a t the A P activity o f c o n t a m i n a t i n g a s t r o c y t e s was unlikely to have a n y significant influence on o u r results. T h e n e u r o t r o p h i c effect o f b F G F on o u r c u l t u r e d neur o n s was d e t e c t e d b o t h b y A P a s s a y a n d by c o u n t i n g n e u r o n a l cell n u m b e r s . T h e effective c o n c e n t r a t i o n o f b F G F in o u r e x p e r i m e n t s was in g o o d a g r e e m e n t with those r e p o r t e d b y S a i t o [4] a n d W a l i c k [6]. In A P assay, 0.3 ng/ml a n d 1 ng/ml b F G F e n h a n c e d n e u r o n a l survival b y 129% a n d 141% o f c o n t r o l (Table II). In c o u n t i n g assay, e n h a n c e d survival rates were 126% a n d 135% o f c o n t r o l for 0.3 ng/ml a n d 1 ng/ml b F G F , respectively. We s h o w e d t h a t A P activity p e r 103 cells was a l m o s t c o n s t a n t with o r w i t h o u t b F G F w h e n c o m p a r e d in the s a m e e x p e r i m e n t (Table II). W e c a n c o n c l u d e t h a t the n e u r o t r o p h i c effect o f b F G F d e t e c t e d b y A P a s s a y is p r o b a b l y d u e to the i m p r o v e m e n t o f n e u r o n a l survival a n d n o t to the a u g m e n t a t i o n o f A P activity p e r neurons. A P levels h a v e b e e n s h o w n to v a r y u n d e r certain c o n d i tions in o t h e r cell t y p e s a n d culture c o n d i t i o n s . F o r example, A P activity is a m a t u r a t i o n m a r k e r o f m a c r o p h a g e a n d is different a c c o r d i n g to culture ages [14]. So c o n t r o l s s h o u l d be i n c l u d e d in each e x p e r i m e n t to ensure t h a t the e n z y m e levels reflect the a c t u a l cell n u m b e r .
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207 12 Hatten, M.E., Lynch, M., Rydel, R.E., Sanchez, J,, Joseph-Silverstein, J., Moscatelli, D. and Rifkin, D.B., In vitro neurite extension by granule neurons is dependent upon astroglial-derived fibroblast growth factor, Dev. Biol., 125 (1988) 280-289. 13 Cataldo, A.M., Paskevich, P.A., Kominami, E. and Nixon, R.A., Lysosomal hydrolase of different classes are abnormally distributed
in brains of patient with Alzheimer disease, Proc. Natl. Acad. Sci. USA, 88(1991) 10998 11002. 14 Becker, S., Warren, M.K. and Haskill, S., Colony-stimulation |'actor-induced monocyte survival and differentiation into macrophages in serun free culture. J. Immunol., 139 (1987) 3703 3709.