This article was downloaded by: [University of Prince Edward Island] On: 18 November 2014, At: 13:50 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
A Modified Colorimetric MTT Assay Adapted for Primary Cultured Hepatocytes: Application to Proliferation and Cytotoxicity Assays ab
a
ac
a
a
Masato Oka , Sumio Maeda , Nobumitsu Koga , Katsuya Kato & Takao Saito a
Government Industrial Research Institute, Nagoya, Hirate-cho 1-1, Kita-ku, Nagoya 462, Japan b
On the leave from Gifu Prefectural Industrial Research Technical Center (Gifu, Japan).
c
On leave from Maekawa MFG. Co., Ltd. (Tokyo, Japan). Published online: 12 Jun 2014.
To cite this article: Masato Oka, Sumio Maeda, Nobumitsu Koga, Katsuya Kato & Takao Saito (1992) A Modified Colorimetric MTT Assay Adapted for Primary Cultured Hepatocytes: Application to Proliferation and Cytotoxicity Assays, Bioscience, Biotechnology, and Biochemistry, 56:9, 1472-1473, DOI: 10.1271/bbb.56.1472 To link to this article: http://dx.doi.org/10.1271/bbb.56.1472
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (9), 1472-1473, 1992
Note
A Modified Colorimetric MTT Assay Adapted for Primary Cultured Hepatocytes: Application to Proliferation and Cytotoxicity Assays Masato OKA, * Sumio MAEDA, t Nobumitsu KOGA, ** KatsuyaKATo, and Takao SAITO
Downloaded by [University of Prince Edward Island] at 13:50 18 November 2014
Government Industrial Research Institute, Nagoya, Hirate-cho 1-1, Kita-ku, Nagoya 462, Japan Received February 7, 1992
Primary culture ofhepatocytes has been widely used as an in vitro system for studying multiple liver functions. In this culture, however, correct counting of the cells is difficult. This is because once isolated cells attach to a culture base, their cell-to-cell interactions become very tight, so that they cannot be dispersed perfectly by the conventional techniques (e.g., trypsin/EDTA treatment).1.2) To resolve this problem, we tried to apply a colorimetric MTT assay to primary cultured hepatocytes. The MTT assay is a rapid colorimetric microtiter assay which was originally developed to measure the living cell number of lymphocytes. 3) This method is based on the selective ability of mitochondrial dehydrogenases in living cells to reduce the yellow soluble salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to a purple insoluble formazan precipitate. The number of viable cells is, therefore, 200 III of primary cultured hepatocytes in WE medium
•
remove the medium by aspiration
t
add 110 III of WE medium containig 10 III of MTT solution
Iincubate for 4 hr at 37°C add 100 Jl of acidifed isopropanol
remove thte medium by aspiration
t
wasJ once with
measure O.D.(595-655nm) lorlginal protocol
PBS
t
I
add 20 III of 0.25% trypsin
measurable as the concentration of the MTT reaction product in a spectrophotometer. The advantages of this assay system are its precision and rapidity. In addition, the absence of a requirement for dispersion of cultured cells to measure the cell number is suitable for primary cultured hepatocytes. In this report, we modified the MTT assay to adapt it for primary cultured hepatocytes, and showed that the modified method worked for estimating the cell number of primary cultured hepatocytes. We also showed that the modified MTT assay was usable for hepatocyte proliferation assays and in vitro cytotoxicity assays using primary cultured hepatocytes. First, we attempted to use the MTT assay for primary cultured hepatocytes following the original protocol described by Mosmann 3 ) (Fig. 1). As shown in Fig. 2, although a roughly linear relationship between optical density and cell number was obtained, this protocol seemed to work incompletely because the MTT formazan crystals formed in the hepatocytes were tightly attached to the well bottoms of the microtiter plate and they could not be dissolved perfectly even by thorough mixing. Therefore we made slight modifications in the final crystal-dissolving step (Fig. 1). Instead of direct addition of acidified isopropanol to the medium including the crystals, (1) removal of the medium, (2) washing with PBS, (3) treatment by trypsin, and (4) dissolving the crystals in acidified isopropanol, were done. By these modifications, complete solubilization of the crystals were observed. Moreover, this resulted in a significant increase in sensitivity and a decrease in standard deviation compared to the result using the original method (Fig. 2). There was also a good linear relationship between optical density and cell number from 2,000 to 36,000 cells/well. From these data, we considered the modified MTT assay was practical and useful for estimating the cell number of primary cultured hepatocytes. Next, we tried to use the modified MTT assay in the hepatocytes 1.2
I Incubate for 5 min , at room temp. add 100 III of acidified isopropanol
er:::
U') U')
t
modified protocol
Fig. 1.
..0
0>
0.8
~
0.6
e 'iii
I
original method
r:::
Q)
"C
0.4
'i
Original and Modified Protocols for MTT Assay.
Primary hepatocytes were isolated from adult Wistar rats by Seglen's collagenase perfusion method. 5 ) The living cell number of isolated hepatocytes in suspension were counted by the trypan blue exclusion method using a hemocytometer. Then hepatocytes were seeded into a flat-bottomed 96-well tissue culture plate coated with type I collagen, and cultured at 3TC in a 5% CO 2 atmosphere. The medium for hepatocyte cultures was WE with 10% fetal bovine serum, 100mM dexamethasone, and 100 mM insulin. MTT solution was prepared at 5 mg/ml in PBS (pH 7.2). MTT reactions were done by the protocols shown in the figure. Acidified isopropanol in the figure is isopropanol which contains 0.04 N HCI. In either the original or modified protocol, the last formazan-dissolving step was done by repeated pipetting with a multichannel pipettor. The optical densities of the formazan solutions in wells were measured by a Bio-Rad model 3550 EIA reader. Although ODS70-630 had been used in the original report,3) we found that OD S95 _ 655 could replace OD570-630 with practically no problem (data not shown).
•
(.C)
measure O.D.(595-655nm)
I
O modified method
1.0
.~
c..
0.2
0
O.Ol'llJ---'--.L---'--L.--'----L-"""--.I
o
10000
20000
30000
40000
cell number (cell/well)
Fig. 2.
Comparison between Original and Modified MTT Methods.
Isolated hepatocytes were counted by the method shown in the legend of Fig. 1 and seeded at various cell numbers between 0 and 36,000cells/well. Then they were cultured for 4 hr to allow the cells in suspension to attach on the culture plate. Other details about cell culture and MTT assay followed the protocols and the legend of Fig. 1. The points and bars on graph represent the means and standard deviations of 4 replicates, respectively.
* **
On leave from Gifu Prefectural Industrial Research Technical Center (Gifu, Japan). On leave from Maekawa MFG. Co., Ltd. (Tokyo, Japan). t To whom correspondence should be addressed. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline: WE, Williams' medium E; MEM, Eagle's minimum essential medium.
NII-Electronic Library Service
1473
A Modified MTT Assay Adapted for Primary Cultured Hepatocytes
2r----------------------.
1.0
r--........
:-IHWt:~..,.IIII,H,I----,
.
,, ,, ,, ,
.'
~
I'I'
",• ", , ,, ,, ,, ,, ,, ,
~
.~ 0.5
1\
'.
:J
• I
en
,
o.~ 0 -3
o
1 0. 1
1 00
1 01
102
compounds concentrations (mg/ml) control
EGF Spleen soluble matrix fraction
Fig. 3. Hepatocyte Proliferation Assay Using the Modified MTT Method.
Downloaded by [University of Prince Edward Island] at 13:50 18 November 2014
1 0. 2
1
Isolated hepatocytes were seeded at 10,000 cells/well, and cultured for 4 hr. Then medi um was replaced by 200 pi of fresh medium containing EO F (100 ng/ml) or bovine spleen soluble matrix fraction (225 pg-protein/ml). After subsequent culture for 68 hr, the MTT assay was done as described in Fig. 1. The values of proliferation rate were the relative values to the cell number of the control as I. The cell number of each sample was calculated from the standard curve of Fig. 2. The mean and standard deviation of each sample were deduced from 4 replicates.
proliferation assay and in vitro cytotoxicity assay using primary cultured hepatocytes. Figure 3 shows the result of a hepatocyte proliferation assay using epidermal growth factor (EGF)1.2) and spleen soluble matrix fraction,4) which had been reported to show hepatocyte proliferation activity. In this assay system, significant mitogenic effects were detected in the samples containing either EGF or spleen soluble matrix fraction compared to the control sample (not containing mitogens). This indicates that the modified MTT assay is usable as a sensitive assay system for hepatocytes proliferation. Figure 4 shows the result of an in vitro cytotoxicity assay using primary cultured hepatocytes. In this assay three hepatotoxic compounds, carbon tetrachloride, N-nitrosodimethylamine, and D-galactosamine, 1,2) were used as test chemicals. For all three compounds, this assay system was able to detect the toxic effects on hepatocytes in response to increasing concentrations of the compounds. When using the fibroblast L929, on the other hand, little or lower toxic effects were observed. These results indicate that the modified MTT assay is also available for a hepatocyte-specific cytotoxicity assay showing fine sensitivity. As described above, the modified MTT assay worked successfully for estimating the cell number of primary cultured hepatocytes. Although the colorimetric MTT assay is an indirect method in regard to counting the cell number, there seem to be few problems if the assay is done with proper control(s). In the hepatocyte proliferation assay, eHJ thymidine or [ 125 1J iododeoxyuridine incorporation have usually been used. 1.2) One of the
Fig. 4. In Vitro Cytotoxicity Assay Using Primary Cultured Hepatocytes and the Modified MTT Method. Isolated hepatocytes and L929 cells were seeded at 30,OOOcells/well, and cultured for 4 hr. Then medium was replaced by 200 pI of fresh medium containing various concentrations of carbon tetrachloride, N-nitrosodimethylamine, or Dgalactosamine. After subsequent culture for 68 hr, the MTT assay was done as described in Fig. I. For L929 cell cultures, MEM with 10% fetal bovine serum was used. The values of the survival rate were the relative values to the optical density of the control as I. Each point on the graph shows the mean of 4 replicates. Symbols used: hepatocytes; -----, L929 cells; . , carbon tetrachloride; . , N-nitrosodimethylamine; . , D-galactosamine.
advantages of the MTT assay is that it is done without using any radioisotopes. In this respect, the modified MTT assay is a useful alternative to the conventional radioisotope incorporation assay. The cytotoxicity assay using primary cultured hepatocytes is expected to be a promising system for evaluating the toxic effects of chemicals on the human body. This is because the liver (and the hepatocyte) is a principal target for the toxic chemicals in the animal body, and the modified MTT assay is capable of detecting a slight change of hepatocyte viability as shown above. Moreover, the ability of this system to handle large numbers of samples is particularly suitable for the evaluation of combinational effects of mUltiple toxic compounds or the screening of toxic matters. We are now trying to apply this system to the evaluation of the toxic effects of environmental pollution.
References 1)
2) 3) 4) 5)
A. Guillouzo and C. Guguen-Guillouzo, "Isolated and Cultured Hepatocytes," John Libbey EurotextjlNSERM, London and Paris, 1986. T. Nakamura, "Syodai Baiyou Kansaibou Jikkenhou" (in Japanese), Gakkai Shupp an Center, Tokyo, 1987. T. Mosmann, J. Immuno. Methods, 65, 55-63 (1983). T. Suzuki, N. Koga, T. Imamura, and Y. Mitsui, Biochem. Biophys. Res. Commun., 153, 1123-1128 (1988). P. O. Seglen, Methods Cell Bioi., 13, 29-83 (1976).
NII-Electronic Library Service
Bioscience, Biotechnology, and Biochemistry Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbbb20
A Modified Colorimetric MTT Assay Adapted for Primary Cultured Hepatocytes: Application to Proliferation and Cytotoxicity Assays ab
a
ac
a
a
Masato Oka , Sumio Maeda , Nobumitsu Koga , Katsuya Kato & Takao Saito a
Government Industrial Research Institute, Nagoya, Hirate-cho 1-1, Kita-ku, Nagoya 462, Japan b
On the leave from Gifu Prefectural Industrial Research Technical Center (Gifu, Japan).
c
On leave from Maekawa MFG. Co., Ltd. (Tokyo, Japan). Published online: 12 Jun 2014.
To cite this article: Masato Oka, Sumio Maeda, Nobumitsu Koga, Katsuya Kato & Takao Saito (1992) A Modified Colorimetric MTT Assay Adapted for Primary Cultured Hepatocytes: Application to Proliferation and Cytotoxicity Assays, Bioscience, Biotechnology, and Biochemistry, 56:9, 1472-1473, DOI: 10.1271/bbb.56.1472 To link to this article: http://dx.doi.org/10.1271/bbb.56.1472
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Biosci. Biotech. Biochem., 56 (9), 1472-1473, 1992
Note
A Modified Colorimetric MTT Assay Adapted for Primary Cultured Hepatocytes: Application to Proliferation and Cytotoxicity Assays Masato OKA, * Sumio MAEDA, t Nobumitsu KOGA, ** KatsuyaKATo, and Takao SAITO
Downloaded by [University of Prince Edward Island] at 13:50 18 November 2014
Government Industrial Research Institute, Nagoya, Hirate-cho 1-1, Kita-ku, Nagoya 462, Japan Received February 7, 1992
Primary culture ofhepatocytes has been widely used as an in vitro system for studying multiple liver functions. In this culture, however, correct counting of the cells is difficult. This is because once isolated cells attach to a culture base, their cell-to-cell interactions become very tight, so that they cannot be dispersed perfectly by the conventional techniques (e.g., trypsin/EDTA treatment).1.2) To resolve this problem, we tried to apply a colorimetric MTT assay to primary cultured hepatocytes. The MTT assay is a rapid colorimetric microtiter assay which was originally developed to measure the living cell number of lymphocytes. 3) This method is based on the selective ability of mitochondrial dehydrogenases in living cells to reduce the yellow soluble salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) to a purple insoluble formazan precipitate. The number of viable cells is, therefore, 200 III of primary cultured hepatocytes in WE medium
•
remove the medium by aspiration
t
add 110 III of WE medium containig 10 III of MTT solution
Iincubate for 4 hr at 37°C add 100 Jl of acidifed isopropanol
remove thte medium by aspiration
t
wasJ once with
measure O.D.(595-655nm) lorlginal protocol
PBS
t
I
add 20 III of 0.25% trypsin
measurable as the concentration of the MTT reaction product in a spectrophotometer. The advantages of this assay system are its precision and rapidity. In addition, the absence of a requirement for dispersion of cultured cells to measure the cell number is suitable for primary cultured hepatocytes. In this report, we modified the MTT assay to adapt it for primary cultured hepatocytes, and showed that the modified method worked for estimating the cell number of primary cultured hepatocytes. We also showed that the modified MTT assay was usable for hepatocyte proliferation assays and in vitro cytotoxicity assays using primary cultured hepatocytes. First, we attempted to use the MTT assay for primary cultured hepatocytes following the original protocol described by Mosmann 3 ) (Fig. 1). As shown in Fig. 2, although a roughly linear relationship between optical density and cell number was obtained, this protocol seemed to work incompletely because the MTT formazan crystals formed in the hepatocytes were tightly attached to the well bottoms of the microtiter plate and they could not be dissolved perfectly even by thorough mixing. Therefore we made slight modifications in the final crystal-dissolving step (Fig. 1). Instead of direct addition of acidified isopropanol to the medium including the crystals, (1) removal of the medium, (2) washing with PBS, (3) treatment by trypsin, and (4) dissolving the crystals in acidified isopropanol, were done. By these modifications, complete solubilization of the crystals were observed. Moreover, this resulted in a significant increase in sensitivity and a decrease in standard deviation compared to the result using the original method (Fig. 2). There was also a good linear relationship between optical density and cell number from 2,000 to 36,000 cells/well. From these data, we considered the modified MTT assay was practical and useful for estimating the cell number of primary cultured hepatocytes. Next, we tried to use the modified MTT assay in the hepatocytes 1.2
I Incubate for 5 min , at room temp. add 100 III of acidified isopropanol
er:::
U') U')
t
modified protocol
Fig. 1.
..0
0>
0.8
~
0.6
e 'iii
I
original method
r:::
Q)
"C
0.4
'i
Original and Modified Protocols for MTT Assay.
Primary hepatocytes were isolated from adult Wistar rats by Seglen's collagenase perfusion method. 5 ) The living cell number of isolated hepatocytes in suspension were counted by the trypan blue exclusion method using a hemocytometer. Then hepatocytes were seeded into a flat-bottomed 96-well tissue culture plate coated with type I collagen, and cultured at 3TC in a 5% CO 2 atmosphere. The medium for hepatocyte cultures was WE with 10% fetal bovine serum, 100mM dexamethasone, and 100 mM insulin. MTT solution was prepared at 5 mg/ml in PBS (pH 7.2). MTT reactions were done by the protocols shown in the figure. Acidified isopropanol in the figure is isopropanol which contains 0.04 N HCI. In either the original or modified protocol, the last formazan-dissolving step was done by repeated pipetting with a multichannel pipettor. The optical densities of the formazan solutions in wells were measured by a Bio-Rad model 3550 EIA reader. Although ODS70-630 had been used in the original report,3) we found that OD S95 _ 655 could replace OD570-630 with practically no problem (data not shown).
•
(.C)
measure O.D.(595-655nm)
I
O modified method
1.0
.~
c..
0.2
0
O.Ol'llJ---'--.L---'--L.--'----L-"""--.I
o
10000
20000
30000
40000
cell number (cell/well)
Fig. 2.
Comparison between Original and Modified MTT Methods.
Isolated hepatocytes were counted by the method shown in the legend of Fig. 1 and seeded at various cell numbers between 0 and 36,000cells/well. Then they were cultured for 4 hr to allow the cells in suspension to attach on the culture plate. Other details about cell culture and MTT assay followed the protocols and the legend of Fig. 1. The points and bars on graph represent the means and standard deviations of 4 replicates, respectively.
* **
On leave from Gifu Prefectural Industrial Research Technical Center (Gifu, Japan). On leave from Maekawa MFG. Co., Ltd. (Tokyo, Japan). t To whom correspondence should be addressed. Abbreviations: MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; PBS, phosphate-buffered saline: WE, Williams' medium E; MEM, Eagle's minimum essential medium.
NII-Electronic Library Service
1473
A Modified MTT Assay Adapted for Primary Cultured Hepatocytes
2r----------------------.
1.0
r--........
:-IHWt:~..,.IIII,H,I----,
.
,, ,, ,, ,
.'
~
I'I'
",• ", , ,, ,, ,, ,, ,, ,
~
.~ 0.5
1\
'.
:J
• I
en
,
o.~ 0 -3
o
1 0. 1
1 00
1 01
102
compounds concentrations (mg/ml) control
EGF Spleen soluble matrix fraction
Fig. 3. Hepatocyte Proliferation Assay Using the Modified MTT Method.
Downloaded by [University of Prince Edward Island] at 13:50 18 November 2014
1 0. 2
1
Isolated hepatocytes were seeded at 10,000 cells/well, and cultured for 4 hr. Then medi um was replaced by 200 pi of fresh medium containing EO F (100 ng/ml) or bovine spleen soluble matrix fraction (225 pg-protein/ml). After subsequent culture for 68 hr, the MTT assay was done as described in Fig. 1. The values of proliferation rate were the relative values to the cell number of the control as I. The cell number of each sample was calculated from the standard curve of Fig. 2. The mean and standard deviation of each sample were deduced from 4 replicates.
proliferation assay and in vitro cytotoxicity assay using primary cultured hepatocytes. Figure 3 shows the result of a hepatocyte proliferation assay using epidermal growth factor (EGF)1.2) and spleen soluble matrix fraction,4) which had been reported to show hepatocyte proliferation activity. In this assay system, significant mitogenic effects were detected in the samples containing either EGF or spleen soluble matrix fraction compared to the control sample (not containing mitogens). This indicates that the modified MTT assay is usable as a sensitive assay system for hepatocytes proliferation. Figure 4 shows the result of an in vitro cytotoxicity assay using primary cultured hepatocytes. In this assay three hepatotoxic compounds, carbon tetrachloride, N-nitrosodimethylamine, and D-galactosamine, 1,2) were used as test chemicals. For all three compounds, this assay system was able to detect the toxic effects on hepatocytes in response to increasing concentrations of the compounds. When using the fibroblast L929, on the other hand, little or lower toxic effects were observed. These results indicate that the modified MTT assay is also available for a hepatocyte-specific cytotoxicity assay showing fine sensitivity. As described above, the modified MTT assay worked successfully for estimating the cell number of primary cultured hepatocytes. Although the colorimetric MTT assay is an indirect method in regard to counting the cell number, there seem to be few problems if the assay is done with proper control(s). In the hepatocyte proliferation assay, eHJ thymidine or [ 125 1J iododeoxyuridine incorporation have usually been used. 1.2) One of the
Fig. 4. In Vitro Cytotoxicity Assay Using Primary Cultured Hepatocytes and the Modified MTT Method. Isolated hepatocytes and L929 cells were seeded at 30,OOOcells/well, and cultured for 4 hr. Then medium was replaced by 200 pI of fresh medium containing various concentrations of carbon tetrachloride, N-nitrosodimethylamine, or Dgalactosamine. After subsequent culture for 68 hr, the MTT assay was done as described in Fig. I. For L929 cell cultures, MEM with 10% fetal bovine serum was used. The values of the survival rate were the relative values to the optical density of the control as I. Each point on the graph shows the mean of 4 replicates. Symbols used: hepatocytes; -----, L929 cells; . , carbon tetrachloride; . , N-nitrosodimethylamine; . , D-galactosamine.
advantages of the MTT assay is that it is done without using any radioisotopes. In this respect, the modified MTT assay is a useful alternative to the conventional radioisotope incorporation assay. The cytotoxicity assay using primary cultured hepatocytes is expected to be a promising system for evaluating the toxic effects of chemicals on the human body. This is because the liver (and the hepatocyte) is a principal target for the toxic chemicals in the animal body, and the modified MTT assay is capable of detecting a slight change of hepatocyte viability as shown above. Moreover, the ability of this system to handle large numbers of samples is particularly suitable for the evaluation of combinational effects of mUltiple toxic compounds or the screening of toxic matters. We are now trying to apply this system to the evaluation of the toxic effects of environmental pollution.
References 1)
2) 3) 4) 5)
A. Guillouzo and C. Guguen-Guillouzo, "Isolated and Cultured Hepatocytes," John Libbey EurotextjlNSERM, London and Paris, 1986. T. Nakamura, "Syodai Baiyou Kansaibou Jikkenhou" (in Japanese), Gakkai Shupp an Center, Tokyo, 1987. T. Mosmann, J. Immuno. Methods, 65, 55-63 (1983). T. Suzuki, N. Koga, T. Imamura, and Y. Mitsui, Biochem. Biophys. Res. Commun., 153, 1123-1128 (1988). P. O. Seglen, Methods Cell Bioi., 13, 29-83 (1976).
NII-Electronic Library Service
