Cytotoxicity Screening Methods Guide

XENOMETRIX
Cytotoxicity Screening
• XTT/MTT
• NR Neutral Red
• SRB Sulforhodamine B
• LDH Lactate dehydrogenase
• CVDE Crystal violet dye elution
• Glucose
Xenometrix GmbH
Gewerbestrasse 25
CH-4123 Allschwil • PAC Acid Phosphatase
Tel ++41· 61· 482 14 34
Fax ++41· 61· 482 20 72 • Combined Kits: 2–4 parameters
email info@xenometrix.ch
www.xenometrix.ch from 1 cellular sample
6560 Gove Court Phone: 513-770-1991 Toll Free: 866-783-3797 FAX: 513-573-9241
Aniara Mason, OH 45040 Web: www.aniara.com Email: info@aniara.com
Table of Contents
1) Introduction
2) Objectives of the IN CYTOTOX Assays
a. Biological Parameters
b. Combined Test Kits
3) Principle of the Assays
4) IN CYTOTOX Test Kits
5) Advantages of the IN CYTOTOX Test Kits
6) Sensitivities of IN CYTOTOX Assays
7) Aniara Product List (IN CYTOTOX and AMES)
8) Technical Specifications
9) Literature
10) CelTox Software
January 2007
IN CYTOTOX Cytotoxicity Test Systems XENOMETRIX
by Endotell
ACID PHOSPHATASE
- Lysosomal activity GLUCOSE
- General physiological
cell state
NEUTRAL RED - Glucose consumption
- Lysosomal activity
- Membrane permeability
MTT / XTT
- Mitochondrial metabolism
LDHe - Respiratory toxicity
- Membrane
permeability
CRYSTAL VIOLET
SULFORHODAMINE B - Cell proliferation
- Cell proliferation - DNA
- Total protein
synthesis rate
1. Introduction
The safety evaluation of compounds such as drugs, cosmetics, food additives,

pesticides and industrial chemicals is growing year by year.
Cytotoxicity assays were among the first in vitro bioassay methods used to predict
toxicity of substances to various tissues. They are widely used for the determination
of cell proliferation, viability and activation. The need for reliable, sensitive and
quantitative assays that would enable analysis of large number of tests in preclinical
research is therefore increasing.
2. Objectives of the IN CYTOTOX Assays
Alternative methods to animal experimentation allow to limit the use of experimental

test animals and to perform screening tests on a greater scale with much lower
quantities of test compound. The alternative methods are the scientific and economic
tool of choice for molecular or cellular high throughput screening.
Xenometrix offers with the IN CYTOTOX products a complete solution for the in vitro
evaluation of tolerance, resistance and recovery of cells in response to
pharmaceutical or chemical compounds. They are based on sensitive and fast high
throughput methods.
IN CYTOTOX test kits can be used for any cell line as well as for spheroids resulting
from human breast or colon cancer.
a. Biological Parameters
The IN CYTOTOX high-throughput test systems allow the measurements of

membrane integrity (LDHe), metabolic activity (GLU), respiratory chain activity
(XTT/MTT), total protein synthesis (SRB), DNA content/cell number (CVDE), and
lysosomal activity (PAC, NR). Studies of agonist and antagonist interactions can be
performed.
The parameters ID50 or IC50 (50% inhibiting dose or concentration), NED (no effect
dose) and IT50 (inhibiting time) can be determined.
.
b. Combined Test Kits
The combined IN CYTOTOX test kits allow to determine up to four metabolic

parameters from the same cellular sample. For example membrane integrity, cellular
metabolism, mitochondrial activity and total protein synthesis or cell proliferation can
be assessed from the same cells. This technical optimization used in the combined
kits allows to increase the relevance of correlation of the tests, to reduce the handling
time, and the amounts of test compound. It allows also to make optimal use of rare
and valuable cell samples such as primary cell cultures.
3. Principle of the Assays
The IN CYTOTOX assays consist of four steps:
- trypsinization of the cells

- transfer to 96-well plates
- test compound incubation
- cytotoxicity assays
Cells are harvested by trypsinisation, counted, diluted and transferred to 96-well

microtiter plates. (Non-adherent cells can be used as well but require more gentle
handling and washing steps). After incubation for at least 24 hrs dilutions of test
compound are added. The cells are exposed to the test compound for the desired
length of time. Cell growth and viability are then measured by using several
cytotoxicity assays.
The test results are measured spectrophotometrically at specific wave lengths. The
analysis of the results can be performed using the CelTox software.
4. IN CYTOTOX Test Kits
One Parameter Kits
XTT and MTT Tetrazolium salt

LDHe Extracellular Lactate Dehydrogenase
NR Neutral Red
PAC Acid Phosphatase
SRB Sulforhodamine B
GLU Glucose
CVDE Crystal Violet Dye Elution
Multiple Parameter Kits
XTT - CVDE
XTT - PAC
XTT - SRB
NR - CVDE
NR - SRB
SRB - CVDE
LDHe - XTT
LDHe - XTT - NR
LDHe - XTT - SRB
XTT - SRB - CVDE
XTT - NR - SRB
LDHe - GLU - XTT - PAC
LDHe - GLU - XTT - SRB
LDHe - XTT - NR - SRB
5. Advantages of the IN CYTOTOX Test Kits
- Complete range of cytotoxicity markers
- High throughput screening methods
- Easy handling
- Single or multiple parameters from one cellular sample
- Reduced amount of test chemicals with the multiple parameter test kits
- Specific CelTox software available
- Customized reporting format
- Swiss manufacture
- Dedicated customer support by experienced staff
6. Sensitivities of In Cytotox Assays
LDHe XTT/MTT
1.8 2
1.6 Medium 1
1.8
1.6
OD480-OD690
1.4 40000
1.4
20000 1.2
1.2 XTT
10000 1
OD340
1 5000 0.8 MTT

0.8 2500 0.6
0.4
0.6 1250
0.2
625
0.4 0
312
Medium 1
Medium 2
40000
20000
10000
5000
2500
1250
625
312
156
78
0.2
156
0 78
0 10 20 30 40 50 60 70 Medium 2
Time (min.) Cells per well
SRB NR
2.0000 0.8
1.8000 0.7
1.6000
0.6
OD540-OD690
1.4000 OD540-OD690
1.2000 0.5
1.0000 0.4
0.8000
0.3
0.6000
0.2
0.4000
0.2000 0.1
0.0000 0
Medium 1
40000
20000
10000
5000
2500
1250
625
312
156
78
Medium 2
Medium 1
40000
20000
10000
5000
2500
1250
625
312
156
78
Medium 2
Cells per well Cells per well
PAC CVDE
1.8000 1
1.6000 0.9
1.4000 0.8
OD405-OD690
OD540-OD690
1.2000 0.7
0.6
1.0000
0.5
0.8000
0.4
0.6000
0.3
0.4000 0.2
0.2000 0.1
0.0000 0
Medium 1
40000
20000
10000
5000
2500
1250
625
312
156
78
Medium 2
40000
20000
10000
Medium 1
5000
2500
1250
625
312
156
78
Medium 2
Cells per well Cells per well
Sensitivity: smallest number of cells per microtiter well giving a significant (p<0.05) signal above medium
control.
Culture conditions: L929 mouse fibroblasts seeded in triplicates at the cell numbers indicated and grown
overnight. Actual sensitivity may vary with cell type and culture conditions.
LDHe: 1250
XTT: 78
MTT: 1250
SRB: 156
NR: 625
PAC: 625
CVDE: 2500
7. Aniara Product List
Product Art.No.
Single Parameter Kits
CVDE Cristal Violet Dye Elution

300 tests AKCV 96.300
1200 tests AKCV 96.1200
GLU Glucose
400 tests AKGLU 96.400
1200 tests AKGLU 96.1200
LDHe Extracellular Lactate Dehydrogenase

300 tests AKLE 96.300
1200 tests AKLE 96.1200
2400 tests AKLE 96.2400
4800 tests AKLE 96.4800
MTT Diphenyltetrazolium bromide

300 tests AKMT 96.300
1200 tests AKMT 96.1200
NR Neutral Red
300 tests AKRN 96.300
1200 tests AKRN 96.1200
PAC Acid Phosphatase

300 tests AKPA 96.300
1200 tests AKPA 96.1200
SRB Sulforhodamine B
300 tests AKSR 96.300
1200 tests AKSR 96.1200
XTT Tetrazolium hydroxide

300 tests AKXT 96.300
1200 tests AKXT 96.1200
2400 tests AKXT 96.2400
4800 tests AKXT 96.2400
Product Art.No.
Multiple Parameter Kits
NR - CVDE
2 x 300 tests AKRCV 96.300
2 x 1200 tests AKRCV 96.1200
NR - SRB
2 x 300 tests AKRSR 96.300
2 x 1200 tests AKRSR 96.1200
SRB - CVDE
2 x 300 tests AKSRCV 96.300
2 x 1200 tests AKSRCV 96.1200
XTT - CVDE
2 x 300 tests AKXCV 96.300
2 x 1200 tests without microplates AKXCV 96.1200
LDHe - XTT
2 x 300 tests AKLEX 96.300
2 x 1200 tests AKLEX 96.1200
XTT - PAC
2 x 300 tests AKXPAC 96.300
2 x 1200 tests AKXPAC 96.1200
XTT - SRB
2 x 300 tests AKXSR 96.300
2 x 1200 tests AKXSR 96.1200
LDHe - XTT - NR
3 x 300 tests AKLEXR 96.300
3 x 1200 tests AKLEXR 96.1200
LDHe - XTT - SRB

3 x 300 tests AKLEXSR 96.300
3 x 1200 tests AKLEXSR 96.1200
Product Art.No.
XTT - SRB - CVDE

3 x 300 tests AKXTSCV 96.300
3 x 1200 tests AKXTSCV 96.1200
XTT - NR - SRB
3 x 300 tests AKXTRS 96.300
3 x 1200 tests AKXTRS 96.1200

4 x 300 tests AKLGXP 96.300
4 x 1200 tests AKLGXP 96.1200
LDHe - GLU - XTT - SRB

4 x 300 tests AKLGXS 96.300
4 x 1200 tests AKLGXS 96.1200
PAN I : LDHe - XTT - NR - SRB

4 x 300 tests APAN I 96.300
4 x 1200 tests APAN I 96.1200
Celtox Software AICT.Celtox
Plastic Ware (microplates and reservoirs) are available on request:

info@aniara.com
Product Art.No.
All Ames MPF kits are available with semisolid strains which can be
transported at room temperature, but have to be stored at -70 C,
or at -20 C for 6 months.
*The experimental design (number of doses, placement of controls

on the plates) may be customized according to the personal needs.
Ames MPF 98/100 - 10 Samples Kit ATAG-98/100-10

8 Vials semisolid TA100
8 Vials Ampicillin
Growth media, Exposure Media and Indicator Media
Reagents provided are sufficient to analyze in up to 8 experiments
at least 10 compounds in triplicate at 6 concentrations, as well as
zero dose and positive controls*.
Ames MPF 98/100 - 10 Samples Kit - Rat Liver S9 ATAG-98/100-10-S9

same components as in Ames MPF 98/100 - 10, plus:
lyophilized rat liver S9
Ames MPF 98/100 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAG-98/100-10-C

Positive Controls: 2-NF, 2-AA, 4-NQO
Ames MPF 98/100 - 1 Sample Kit ATAG-98/100-1

1 Vial semisolid TA98
1 Vial Ampicillin
Growth Media, Exposure Media and Indicator Media
Reagents provided are sufficient to perform 1 sample in triplicate
or 3 samples if performed without replicate at 6 concentrations,
as well as zero dose and positive controls*.
Ames MPF 98/100 - 1 Sample Kit - Rat Liver S9 ATAG-98/100-1-S9

Ames MPF 98/100 - 1 Sample Kit - Rat Liver S9 - Pos.Control ATAG-98/100-1-C

Positive Controls: 2-NF, 2-AA, 4-NQO
Product Art.No.
Ames MPF 98/100/1535/1537 - 10 Samples Kit ATAG-981003537-10

10 Vials Ampicillin
All reagents provided are sufficient to analyze in up to 10
experiments at least 10 compounds with all four strains in triplicate
at 6 concentrations, as well as zero doses and positive controls*.
Ames MPF 98/100/1535/1537 - 10 Samples Kit - Rat Liver S9 ATAG-981003537-10-S9

same components as in Ames MPF 98/100/1535/1537 - 10, plus:
Ames MPF 98/100/1535/1537 - 10 Samples Kit - S9 - Pos.Control ATAG-981003537-10-C

Positive Controls: 2-NF, 2-AA, 9-AA, 4-NQO, N4-ACT
Ames MPF 98/100/1535/1537 - 1 Sample Kit ATAG-981003537-1

1 Vial Ampicillin
All reagents provided are sufficient to perform 1 sample in
triplicate or 3 samples if performed without replicate at 6
concentrations, zero dose and positive controls*.
Ames MPF 98/100/1535/1537 - 1 Sample Kit - Rat Liver S9 ATAG-981003537-1-S9

Ames MPF 98/100/1535/1537 - 1 Sample Kit - S9 - Pos.Control ATAG-981003537-1-C

Product Art.No.
Ames MPF PENTA I E.coli Combination - 10 Samples Kit

AC10-512
10 Vials semisolid E.Coli uvrA or pKM101
10 Vials Ampicillin
experiments at least 10 compounds with all four strains in triplicate
at 6 concentrations, as well as zero doses and positive controls*.

Ames MPF PENTA I E.coli Combination - 10 Samples Kit - Rat Liver S9
AC10-512-S1
same components as in Ames MPF 98/100/1535/1537/E.coli - 10, plus: lyophilized
rat liver S9

Ames MPF PENTA I E.coli Combination - 10 Samples Kit - S9 - Pos.Con. AC10-512-S1-P
rat liver S9
Ames MPF PENTA I E.coli- Combination - 1 Sample Kit

AC01-512
1 Vial semisolid E.Coli uvrA or pKM101
1 Vial Ampicillin

Ames MPF PENTA I E.coli Combination - 1 Sample Kit - Rat Liver S9
AC01-512-S1
rat liver S9

Ames MPF PENTA I E.coli Combination - 1 Sample Kit - S9 - Pos.Con. AC01-512-S1-P
rat liver S9
Ames MPF 98 - 10 Samples Kit ATAG-98-10

8 Vials Ampicillin
experiments at least 10 compounds in triplicate at 6 concentrations
Product Art.No.
Ames MPF 98 - 10 Samples Kit - Rat Liver S9 ATAG-98-10-S9

same components as in Ames MPF 98 - 10, plus:
Ames MPF 98 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAG-98-10-C

Positive Controls: 2-NF, 2-AA
Ames MPF 98 - 1 Sample Kit ATAG-98-1

1 Vial Ampicillin
Ames MPF 98 - 1 Sample Kit - Rat Liver S9 ATAG-98-1-S9

Ames MPF 98 - 1 Sample Kit - Rat Liver S9 - Pos.Control ATAG-98-1-C

Positive Controls: 2-NF, 2-AA

8 Vials Ampicillin
experiments at least 10 compounds in triplicate at 6
concentrations as well as zero dose and positive controls*.


Positive Controls: 4-NQO, 2-AA
Product Art.No.

1 Vial Ampicillin



experiments at least 10 compounds in triplicate at 6 concentrations


Positive Controls: N4-ACT, 2-AA



Positive Controls: N4-ACT, 2-AA
Product Art.No.



Positive Controls: 9-AACR, 2-AA



Positive Controls: 9-AACR, 2-AA
Ames MPF E.Coli uvrA - 10 Samples Kit AECG-uvrA-10

8 Vials semisolid E.Coli uvrA
Ames MPF E.Coli uvrA - 10 Samples Kit - Rat Liver S9 AECG-uvrA-10-S9

same components as in Ames MPF E.Coli uvrA-10, plus:
Ames MPF E.Coli uvrA - 10 Samples Kit - Rat Liver S9 - Pos.Contr. AECG-uvrA-10-C
Product Art.No.
Ames MPF E.Coli uvrA - 1 Sample Kit AECG-uvrA-1

Ames MPF E.Coli uvrA - 1 Sample Kit - Rat Liver S9 AECG-uvrA-1-S9

Ames MPF E.Coli uvrA - 1 Sample Kit - Rat Liver S9 - Pos.Contr. AECG-uvrA-1-C
Ames MPF E.Coli pKM - 10 Samples Kit AECG-pKM-10

8 Vials semisolid E.Coli pKM101
Ames MPF E.Coli pKM - 10 Samples Kit - Rat Liver S9 AECG-pKM-10-S9

same components as in Ames MPF E.Coli pKM-10, plus:
Ames MPF E.Coli pKM - 10 Samples Kit - Rat Liver S9 - Pos.Contr. AECG-pKM-10-C
Ames MPF E.Coli pKM - 1 Sample Kit AECG-pKM-1

1 Vial semisolid E.Coli pKM101
Ames MPF E.Coli pKM - 1 Sample Kit - Rat Liver S9 AECG-pKM-1-S9

Ames MPF E.Coli pKM - 1 Sample Kit - Rat Liver S9 - Pos.Contr. AECG-pKM-1-C
Product Art.No.
Ames MPF E.Coli Combo - 10 Samples Kit AECG-COM-10

8 Vials semisolid E.Coli pKM101
Ames MPF E.Coli Combo - 10 Samples Kit - Rat Liver S9 AECG-COM-10-S9

same components as in Ames MPF E.Coli Combo-10, plus:
Ames MPF E.Coli Combo - 10 Samples Kit - Rat Liver S9 - Pos.Contr. AECG-COM-10-C
Ames MPF E.Coli Combo - 1 Sample Kit AECG-COM-1

1 Vial semisolid E.Coli uvrA
1 Vial semisolid E.Coli pKM101
Ames MPF E.Coli Combo - 1 Sample Kit - Rat Liver S9 AECG-COM-1-S9

Ames MPF E.Coli Combo - 1 Sample Kit - Rat Liver S9 - Pos.Contr. AECG-COM-1-C
Product Art.No.
All Ames MPF kits are also available in the same kit configuration but
with frozen strains to be transported on dry ice and stored at -70 C,
or at -20 C for 6 months.
Ames MPF 98/100 - 10 Samples Kit

Ames MPF 98/100 - 10 Samples Kit - Rat Liver S9 ATAM-98/100-10
Ames MPF 98/100 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAM-98/100-10-S9
ATAM-98/100-10-C
Ames MPF 98/100 - 1 Sample Kit
Ames MPF 98/100 - 1 Sample Kit - Rat Liver S9 ATAM-98/100-1
Ames MPF 98/100 - 1 Sample Kit - Rat Liver S9 - Pos.Control ATAM-98/100-1-S9
ATAM-98/100-1-C
Ames MPF 98/100/1535/1537 - 10 Samples Kit
Ames MPF 98/100/1535/1537 - 10 Samples Kit - Rat Liver S9 ATAM-981003537-10
Ames MPF 98/100/1535/1537 - 10 Samples Kit - S9 - Pos.Control ATAM-981003537-10-S9
ATAM-981003537-10-C
Ames MPF 98/100/1535/1537 - 1 Sample Kit
Ames MPF 98/100/1535/1537 - 1 Sample Kit - Rat Liver S9 ATAM-981003537-1
Ames MPF 98/100/1535/1537 - 1 Sample Kit - S9 - Pos.Control ATAM-981003537-1-S9
ATAM-981003537-1-C
Ames MPF PENTA I E.coli Combination - 10 Samples Kit
Ames MPF PENTA I E.coli Combination - 10 Samples Kit - Rat Liver S9 AD10-512
Ames MPF PENTA I E.coli Combination - 10 Samples Kit - S9 - Pos.Con. AD10-512-S1
AD10-512-S1-P
Ames MPF PENTA I E.coli Combination - 1 Sample Kit

AD01-512
Ames MPF PENTA I E.coli Combination - 1 Sample Kit - Rat Liver S9 AD01-512-S1
Ames MPF PENTA I E.coli Combination - 1 Sample Kit - S9 - Pos.Con. AD01-512-S1-P

Ames MPF 98 - 10 Samples Kit ATAM-98-10
Ames MPF 98 - 10 Samples Kit - Rat Liver S9 ATAM-98-10-S9
Ames MPF 98 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAM-98-10-C

Ames MPF 98 - 1 Sample Kit ATAM-98-1
Ames MPF 98 - 1 Sample Kit - Rat Liver S9 ATAM-98-1-S9
Ames MPF 98 - 1 Sample Kit - Rat Liver S9 - Pos.Control ATAM-98-1-C




Product Art.No.


Ames MPF E.Coli uvrA - 10 Samples Kit AECM-uvrA-10

Ames MPF E.Coli uvrA - 10 Samples Kit - Rat Liver S9 AECM-uvrA-10-S9
Ames MPF E.Coli uvrA - 10 Samples Kit - Rat Liver S9 - Pos.Contr. AECM-uvrA-10-C
Ames MPF E.Coli uvrA - 1 Sample Kit AECM-uvrA-1

Ames MPF E.Coli uvrA - 1 Sample Kit - Rat Liver S9 AECM-uvrA-1-S9
Ames MPF E.Coli uvrA - 1 Sample Kit - Rat Liver S9 - Pos.Contr. AECM-uvrA-1-C
Ames MPF E.Coli pKM - 10 Samples Kit AECM-pKM-10

Ames MPF E.Coli pKM - 10 Samples Kit - Rat Liver S9 AECM-pKM-10-S9
Ames MPF E.Coli pKM - 10 Samples Kit - Rat Liver S9 - Pos.Contr. AECM-pKM-10-C
Ames MPF E.Coli pKM - 1 Sample Kit AECM-pKM-1

Ames MPF E.Coli pKM - 1 Sample Kit - Rat Liver S9 AECM-pKM-1-S9
Ames MPF E.Coli pKM - 1 Sample Kit - Rat Liver S9 - Pos.Contr. AECM-pKM-1-C
Ames MPF E.Coli Combo - 10 Samples Kit AECM-COM-10

Ames MPF E.Coli Combo - 10 Samples Kit - Rat Liver S9 AECM-COM-10-S9
Ames MPF E.Coli Combo - 10 Samples Kit - Rat Liver S9 - Pos.Contr. AECM-COM-10-C
Ames MPF E.Coli Combo - 1 Sample Kit AECM-COM-1

Ames MPF E.Coli Combo - 1 Sample Kit - Rat Liver S9 AECM-COM-1-S9
Ames MPF E.Coli Combo - 1 Sample Kit - Rat Liver S9 - Pos.Contr. AECM-COM-1-C
Product Art.No.
Ames MPF Liquid Media – ready to use

Ames Growth Medium (RT) - 50 ml ATAM-GM50
Ames MPF Exposure Medium (RT) - 100 ml ATAM-EM100
Ames MPF Exposure Medium (RT) - 50 ml ATAM-EM50
Ames MPF Reversion Indicator Medium (RT) - 550 ml ATAM-IM550
Ames MPF Reversion Indicator Medium (RT) - 100 ml ATAM-IM100
Ames MPF E.Coli Liquid Media – ready to use

Ames Growth Medium (RT) - 50 ml ATAM-GM50
Ames MPF Exposure Medium (RT) - 100 ml AECM-EM100
Ames MPF Exposure Medium (RT) - 50 ml AECM-EM50
Ames MPF Reversion Indicator Medium (RT) - 550 ml AECM-IM550
Ames MPF Reversion Indicator Medium (RT) - 100 ml AECM-IM100
10 fold concentrated Exposure Medium - 25 ml ATAM-EM25-10X
Ames – Semisolid S. typhimurium Strains

50 ul Semisolid TA98 (RT/-70°C) AGTA-TA98-AG
50 ul Semisolid EC uvraA (RT/-70°C) AGTA-ECuvrA-AG
50 ul Semisolid EC pKM101 (RT/-70°C) AGTA-ECpKM-AG
Ames – Liquid S. typhimurium Strains

50 ul Liquid TA98 (-70°C) AGTA-TA98
50 ul Liquid TAMix (-70°C) AGTA-TAMix
50 ul Liquid EC uvraA (-70°C) AGTA-ECuvrA
50 ul Liquid EC pKM101 (-70°C) AGTA-ECpKM
Ampicillin (-20°C) AGTA-Amp
Rat liver S9
1 ml Aroclor induced lyophilized microsomal rat liver S9 AGTA-S9-LY1-A
2 ml Aroclor induced lyophilized microsomal rat liver S9 AGTA-S9-LY2-A
1 ml Phenobarbital 5/6 Benzoflavone induced rat liver S9 AGTA-S9-LY1-PB

2 ml Phenobarbital 5/6 Benzoflavone induced rat liver S9 AGTA-S9-LY2-PB
Frozen, liquid microsomal rat liver S9 is available on request: info@aniara.com
Product Art.No.
Positive controls
20 ug 2-NF: 2-Nitrofluorene AGTA-2NF-20
100 ug 2-AA: 2-Aminoanthracene AGTA-2AA-100
50 ug 4-NQO: 4-Nitroquinolone AGTA-4NQO-50
100 mg N4-ACT: N4-Aminocytidine AGTA-N4ACT100
1000 ug 9-AACR: 9-Aminoacridine AGTA-9AACR-1000
Plastic Ware
Plastic ware for 1 Sample Kit - Ames MPF 98/100 ATAM-98/100-PW

5 x culture tubes
1 x 96-well chemical plate
4 x 24-well exposure plates
12 x 384-well revertant colony selection plates
5 x reagent reservoirs
Plastic ware for 1 Sample Kit - Ames MPF 100 ATAM-100-PW

3 x culture tubes
1 x 96-well chemical plate
2 x 24-well exposure plates
6 x 384-well revertant colony selection plates
2 x reagent reservoirs
Analytical Services
Ames MPF 98/100 Service

Ames MPF 98/100 Service
Compounds delivered with indication of solvent
6 different concentrations, triplicates, +/- S9
neg. / positive control, report
Ames MPF 98/100/1535/1537 Service

Ames MPF 98/100/1535/1537 Service
Ames MPF 98/100/1535/1537/E.Coli WP2 uvra/pkM101 Service

Ames MPF 98/100/1535/1537/E.Coli WP2 uvra/pkM101 Service
Product Art.No.
Ames II - 10 Samples Kit AGTA-220

8 Vials TA98 (frameshift mutations)
8 Vials TAMix (base pair substitutions)
8 Vials Ampicillin
Incubation Media, Exposure and Indicator Media
Reagents provided are sufficient to analyze in up to 8
individual experiments at least 10 compounds in triplicate at
6 concentrations as well as zero dose and positive controls*.
Ames II - 1 Sample Kit AGTA-225

1 Vial TA98 (frameshift mutations)
1 Vial TAMix (base pair substitutions)
1 Vial Ampicillin
Incubation Media, Exposure and Indicator Media
Reagents provided are sufficient to perform 1 sample in
triplicate ore 3 samples if performed without replicate at 6
Ames II - 10 Samples Kit - Rat Liver S9 AGTA-220-S9

same components as in Ames II -10, plus:
Ames II - 1 Sample Kit - Rat Liver S9 AGTA-225

same components as in Ames II -1, plus:

IN CYTOTOX XENOMETRIX
by Endotell
CVDE
CRYSTAL VIOLET DYE ELUTION
APPLICATIONS
Colorimetric assay for the quantification of nuclear DNA and cell number in response to
pharmaceutical, chemical and environmental compounds and nutrients.
PRINCIPLE
Crystal Violet is a dye that accumulates in the cell nucleus. The fixed dye, measured
photometrically after solubilization, correlates with the nuclear DNA content and thus
with cell number.
BIOLOGICAL PARAMETERS EVALUATION

• IC50 (Inhibitory Concentration 50%)
• Cell proliferation
CVDE staining of mouse L929 cells
0.9000
0.8000
0.7000
0.6000
OD540-OD690
0.5000
0.4000
0.3000
0.2000
0.1000
0.0000
0 5000 10000 15000 20000 25000 30000 35000 40000
Cell per well
TECHNICAL SPECIFICATIONS
Absorbance: - 540 nm
Approximate assay time: - 1 hour
Available kit configurations: - Reagents only

- Reagents and 96-well microplates, sterile
reagent reservoirs
CVDE Kit content: - Wash solution

- Labeling solution
- Solubilization solution
- Instruction manual
CVDE KIT SIZES AND REFERENCE NUMBERS

REFERENCE NO. NUMBER OF TESTS
AKCV96.300 300
AKCV96.1200 1200
Kits with plasticware (microplates and reservoirs) are also available.

Individual reagents and other kit sizes available upon request.
Version 1.5 01/2007

by Endotell
GLU GLUCOSE
APPLICATIONS
Colorimetric assay for the quantification of the cellular metabolism and glucose
consumption in response to pharmaceutical, chemical and environmental compounds,
and nutrients.
PRINCIPLE
This method measures the overall metabolic activity of cells after drug exposure, using
their glucose consumption as a marker. The amount of remaining glucose is measured
spectrophotometrically in the cell culture medium (GOD / POD method). The test is
suitable for kinetic and endpoint studies. Cells are not exposed to any assay reagents
and remain fully viable and available for further testing.
Particularly suitable for cells with high metabolic activity and for extended drug
exposure times.

• Glucose consumption rate
Approximate assay time: - 45 min

reagent reservoirs
GLU Kit content: - Substrate solution

- Enzyme solution
- Glucose standard solution
GLU KIT SIZES AND REFERENCE NUMBERS

AKGLU96.400 400
AKGLU96.1200 1200

Version 1.5 01/2007

by Endotell
LDHe
EXTRACELLULAR LACTATE DEHYDROGENASE
APPLICATIONS
Colorimetric assay for the quantification of membrane integrity and cellular viability in
response to pharmaceutical, chemical and environmental compounds, and nutrients.
PRINCIPLE
Cytotoxicity or cell death is determined by the amount of membrane damage. The
intracellular enzyme Lactate Dehydrogenase (LDH) is released rapidly from damaged
cells into the cell culture supernatant. NADH consumption, measured kinetically in the
cell supernatant, correlates with the amount of released LDH (LDHe). The cell viability
is inversely proportional to the amount of released LDH.
Unlike some other LDHe assays, the In Cytotox LDHe assay measures the oxidation of
NADH to NAD+ and the concurrent reduction of pyruvate to lactate. By providing an
excess of pyruvate in the reaction mixture, the In Cytotox LDHe assay is therefore
insensitive to pyruvate in the culture medium, which can cause product inhibition of the
reverse reaction implemented in other LDHe assays.

• Membrane integrity
• Cell viability
Approximate assay time: - 35 min (1h 10 min if read at RT)

reagent reservoirs
LDHE Kit content: - Reconstitution solution

- Substrate solution
- Activator solution
LDHE KIT SIZES AND REFERENCE NUMBERS

AKLE96.300 300
AKLE96.1200 1200

Version 1.5 01/2007

by Endotell
NR NEUTRAL RED
APPLICATIONS
Colorimetric assay for the quantification of the membrane permeability and lysosomal
activity of cells in response to pharmaceutical, chemical and environmental compounds,
and nutrients.
PRINCIPLE
The neutral red (NR) assay procedure is a cell survival/viability assay based on the
ability of viable cells to incorporate and bind neutral red within lysosomes. It is generally
performed on adherent cells. NR is a weak cationic dye that readily penetrates the cell
membrane and accumulates intracellularly in lysosomes (lysosomal pH < cytoplasmic
pH), where it binds with anionic sites to the lysosomal matrix (G. Griffon, et al. 1995).
Changes of the cell surface or the sensitive lysosomal membrane lead to lysosomal
fragility and other changes that gradually become irreversible. Such alterations brought
about by the action of xenobiotics result in a decreased uptake and binding of NR. It is
thus possible to distinguish between viable, damaged, or dead cells, which is the basis
of the assay.
The quantity of dye incorporated into cells is measured by spectrometry at 540 nm, and
is directly proportional to the number of cells with an intact membrane.
The assay can be used to evaluate cytotoxicity by determination of the IC50 (50%
inhibiting concentration).

• Membrane permeability
• Lysosomal activity
Absorbance: - 540 nm + 690 nm (reference)
Approximate assay time: - 3 hrs 40 min

reagent reservoirs
NR Kit content: - Wash solution

- Labeling solution
- Fixing solution
NR KIT SIZES AND REFERENCE NUMBERS

AKRN96.300 300
AKRN96.1200 1200

Version 1.5 01/2007

by Endotell
PAC ACID PHOSPHATASE

APPLICATIONS
Colorimetric assay for the quantification of the lysosomal metabolism of cells in
PRINCIPLE
Acid Phosphatase (PAC) is a functional marker of the lysosomal compartment. The
enzyme activity is measured by the conversion of p-nitro-phenylphosphate (pNPP) into
p-nitrophenolate (pNP) and correlates with cell number. The reaction product is
determined spectrophotometrically.

Approximate assay time: - 2 hr 20 min

reagent reservoirs
PAC Kit content: - Wash solution

- Substrate solution
- Lysis solution
- Stop solution
PAC KIT SIZES AND REFERENCE NUMBERS

AKPAC96.300 300
AKPAC96.1200 1200

Version 1.5 01/2007

by Endotell
SRB SULFORHODAMINE B
APPLICATIONS
Colorimetric assay for the quantification of the total protein synthesis rate of cells in
PRINCIPLE
Cell proliferation, measured as total protein synthesis, is a very sensitive toxicology
marker. Sulforhodamine B (SRB) is an anionic dye that binds to proteins
electrostatically. The fixed dye, measured photometrically after solubilization, correlates
with total protein synthesis rate and therefore with cell proliferation.

• Total protein synthesis
(Recommended reference filter: 690 nm)
Approximate assay time: - 2 h 10 min + time to dry the microtiter plate

reagent reservoirs
SRB Kit content: - Wash solution

- Fixing solution
- Labeling solution
- Rinsing solution
SRB KIT SIZES AND REFERENCE NUMBERS

AKSR96.300 300
AKSR96.1200 1200

Version 1.5 01/2007

by Endotell
XTT TETRAZOLIUM HYDROXIDE

MTT DIPHENYLTETRAZOLIUM BROMIDE
APPLICATIONS
Colorimetric assay for the quantification of the mitochondrial metabolism and respiratory
chain activity of cells in response to pharmaceutical, chemical and environmental
compounds, and nutrients.
PRINCIPLE
The tetrazolium salts XTT and MTT can be used to measure the metabolic activity of
viable cells. Tetrazolium salts are reduced to formazan by mitochondrial succinate
dehydrogenase, an enzyme which is only active in cells with an intact metabolism and
respiratory chain. The formazan is quantified photometrically and correlates with the
number of viable cells.
Unlike MTT, the XTT formazan product is water soluble. The XTT assay needs
therefore no solubilization step which allows to obtain results typically after about 3 hr
(depending on incubation length). MTT requires an additional overnight incubation.

• Metabolic cytotoxicity
• Respiratory chain activity

Absorbance: - MTT: 540 nm
- XTT: 480 (optimum) or 450 nm
Approximate assay time: - 3 hrs 10 min (depending on the cell line) +

overnight solubilization step (MTT only)

reagent reservoirs
MTT Kit content: XTT Kit content:

- Substrate solution - Substrate solution
- Solubilization solution - Buffer solution
- Instruction manual - Instruction manual
MTT KIT SIZES AND REFERENCE NUMBERS

AKMT96.300 300
AKMT96.1200 1200
XTT KIT SIZES AND REFERENCE NUMBERS

AKXT96.300 300
AKXT96.1200 1200

Version 1.5 01/2007
Why should I use
XENOMETRIX
by Endotell
XTT instead of MTT
XTT has three main advantages over MTT:
1. XTT has a higher sensitivity and a higher dynamic

range
2. With XTT the reaction results in a soluble

formazan dye. This eliminates a final solubilization
step (unlike MTT) which means less manipulation
and consequently a reduced risk of error (for
instance no air bubbles from SDS or Triton X-100)
3. OD readings with XTT can be taken immediately

after incubation whereas with MTT often an
extended incubation for the solubilization of the
precipitate is required before reading
Comparison XTT versus MTT:
XTT has a higher sensitivity and a higher dynamic range
A)
XTT/MTT, corrected for medium, 15 min
1.8
1.6
OD480/540 - OD 690
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
40000 20000 10000 5000 2500 1250 625 312 156 78
Cell number
MTT XTT
B)
XTT/MTT_2, corrected for medium, 2 hr
1.8
1.6
OD480/540 - OD 690
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
40000 20000 10000 5000 2500 1250 625 312 156 78
Cell number
MTT XTT
Conditions:
L929 cells; 3 hr incubation with XTT or MTT; reading at OD480 (XTT) or OD540 (MTT)
corrected for OD690 .
XTT read immediately after incubation.
MTT read after solubilization with 10% Triton X-100 in Isopropanol / 0.1 N HCl and
mixing, after 15 minutes (A) and after 2 hours at 37°C (B).

by Endotell
LDHe - GLU -
XTT - PAC
APPLICATIONS
Combined colorimetric assays for the quantification of the membrane integrity and the
metabolic, mitochondrial and lysosomal activities of cells in response to pharmaceutical,
chemical and environmental compounds, and nutrients.
PRINCIPLE
This kit allows to measure sequentially four cytotoxicity parameters in one single cell
culture: Membrane integrity (LDHe: Extracellular Lactate Dehydrogenase), metabolic
activity (GLU: Glucose), mitochondrial activity (XTT: Tetrazolium Hydroxide) and
lysosomal activity (PAC: Acid Phosphatase).
Released LDH, a marker for cell damage, is determined kinetically in the medium (by
the rate of NADH consumption). Extracellular glucose concentration (GOD / POD
method) is inversely proportional to the rate of glucose uptake, which is an indicator of
the functional metabolic state of cells. XTT reduction rate to formazan is measured in
the cells and correlates with mitochondrial activity. Intracellular PAC activity is an
indicator for lysosomal activity.

Absorbance: - LDHe: 340 nm
- GLU: 540 nm
- PAC: 405 nm
Approximate assay time (total): - 6 hr 50 min if all steps are done sequentially.
Some incubations may be done in parallel
(LDHe, Glu).

reagent reservoirs
LDHe-GLU-XTT-PAC Kit content: - Buffer solutions

- Enzyme solution
- Stop solutions
- Reconstitution solution
- Substrate solutions
- Wash solution
- Lysis solution

KIT SIZES AND REFERENCE NUMBERS
AKLGXP96.300 4 x 300
AKLGXP96.1200 4 x 1200

Version 1.5 01/2007

by Endotell
LDHe - GLU -
XTT - SRB
APPLICATIONS
Combined colorimetric assays for the quantification of the membrane integrity, cellular
and mitochondrial metabolism and total protein synthesis rate of cells in response to
pharmaceutical, chemical and environmental compounds, and nutrients.
PRINCIPLE
culture: Membrane integrity (LDHe: Extracellular Lactate Dehydrogenase), metabolic
activity (GLU: Glucose), mitochondrial activity (XTT: Tetrazolium Hydroxide) and total
protein synthesis rate (SRB: Sulforhodamine B).
Released LDH, a marker for cell damage, is determined kinetically in the medium
(NADH consumption). Extracellular glucose concentration (GOD / POD method) is
inversely proportional to the rate of glucose uptake, which is an indicator of the
functional metabolic state of cells. XTT is reduced in the cells to formazan by
mitochondrial succinate dehydrogenase. The reduction rate is measured and correlates
with mitochondrial activity. The fixed SRB dye, measured photometrically after
solubilization, correlates with total protein synthesis rate and therefore with cell
proliferation.

- GLU: 540 nm
- SRB: 540 nm
(Recommended reference filter: 690 nm)
Approximate assay time (total): - 6 hr 40 + time to dry the plate (SRB) if all
steps are done sequentially. Some
incubations may be done in parallel (LDHe,
Glu).

reagent reservoirs
LDHe-GLU-XTT-SRB Kit content: - Buffer solutions

- Enzyme solution
- Stop solution
- Labeling solution
- Wash solution
- Rinsing solution
- Fixing solution
LDHe GLU XTT SRB KIT

SIZES AND REFERENCE NUMBERS
AKLGXS96.300 4 x 300
AKLGXS96.1200 4 x 1200

Version 1.5 01/2007

by Endotell
PAN I:
LDHe-XTT-NR-SRB
APPLICATIONS
Combined colorimetric assays for the quantification of the membrane integrity,
mitochondrial metabolism, lysosomal integrity and activity, and total protein synthesis
rate of cells in response to pharmaceutical, chemical and environmental compounds,
and nutrients.
PRINCIPLE
culture: Membrane integrity (LDHe: Extracellular Lactate Dehydrogenase),
mitochondrial activity (XTT: Tetrazolium Hydroxide), lysosomal integrity and activity
(NR: Neutral Red), and total protein synthesis rate (SRB: Sulforhodamine B).
Released LDH, a marker for cell damage, is determined kinetically in the medium
(NADH consumption). XTT is reduced in the cells to formazan by mitochondrial
succinate dehydrogenase. The reduction rate is measured and correlates with
mitochondrial activity. The neutral red (NR) assay procedure is based on the ability of
viable cells to incorporate and bind neutral red within lysosomes. The fixed SRB dye,
measured photometrically after solubilization, correlates with total protein synthesis rate
and therefore with cell proliferation.

- NR: 540 nm
- SRB: 540 nm
(Recommended reference filters: 690 nm)
Approximate assay time (total): - 8 hr 30 + time to dry the plate (SRB) if LDH
incubation is done in parallel with XTT
incubation. SRB reading can also be done the
next morning.

reagent reservoirs
LDHe- XTT-NR-SRB Kit content: - Buffer solutions

- Enzyme solution
- Stop solution
- Labeling solution
- Wash solutions
- Solubilization solutions
- Rinsing solution
- Fixing solutions
PAN I: LDHe-XTT-NR-SRB-KIT
SIZES AND REFERENCE NUMBERS
APAN I 96.300 4 x 300
APAN I 96.1200 4 x 1200

Version 1.5 01/2007
9. Literature
The tetrazolium salts XTT and MTT, two functional markers of mitochondria, are
used to measure the metabolic activity of viable cells. Tetrazolium salts accept
electrons from oxidized substrates which results in their reduction to a colored
formazan product. The amount of formazan produced is proportional to the number
of viable cells present. In contrast to MTT, the formazan product yielded by XTT is
water soluble (1) (2) (3) (4) (5) (7).
Neutral Red is accumulated in the lysosomes of viable, uninjured cells. Well known
chemical carcinogens have been used to demonstrate the utility of the Neutral Red
assay for detecting cytotoxic effects with primary rat hepatocytes. The method is
rapid, easy to perform and suitable for handling large numbers of cultures
simultaneously. Fish hepatoma cells have been used in a study to evaluate the acute
toxicities of alkylbenzenes, phthalate diesters, pesticides and metabolism-mediated
benzopyrenes (6) (8) (9) (19).
Lactate dehydrogenase release is an indicator of cellular damage of many cell

lines, especially during stress. The lactate dehydrogenase assay is used for the
quantification of the membrane integrity and cellular viability of adherent and non-
adherent cells in response to pharmaceutical and chemical compounds (10) (11).
The Sulforhodamine B assay (SRB) is based on the staining of cellular proteins.

The quantification of total protein synthesis rate of adherent or non-adherent cells in
response to chemical compounds are determined. Studies using lung cancer, colon
cancer and breast cancer cell lines, as well as six different human ovarian tumor cell
lines have been performed. The use of the SRB protein staining for in vitro
chemosensitivity testing has been adopted by the US National Cancer Institute
It has been shown, that the SRB assay is simpler, faster and more sensitive than the
MTT assay, provides better linearity with cell number, permits the use of saturating
dye concentrations and is independent of intermediary metabolism (12) (13) (14)(15).
Acid Phosphatase is a functional marker of the lysosomal compartment. It can be

used to assess the integrity of lysosomes and thus to assess the toxicity of
compounds. The In Cytotox Acid Phosphatase (PAC) assay offers an alternative and
extension of other cytotoxicity assays and is particularly suitable to detect with a high
sensitivity substances interfering with the function and integrity of lysosomes. The
assay correlates linearly over a wide range with cell number and has been tested
with several different cell lines. It is simple to perform and can be read after 2 hrs (6).
References
(1) N.W. Roehm, G.H. Rodgers, S.M. Hatfield, and A.L. Glasebrook, An improved
colorimetric assay for cell proliferation and viability utilizing the tetrazolium salt XTT,
Journal of Immunological Methods (1991) 142, 257-265.
(2) A.A. Gedolf, S.C. Mastbergen, R.E.C. Henrar, G.T. Faircloth, Cytotoxicity and
neurocytotoxicity of new marine anticancer agents evaluated using in vitro assays,
Cancer Chemother Pharmacol (1999) 44, 312-318.
(3) D.T. Vistica, P. Skehan, D. Scudiero, A. Monks, A. Pittman and M.R. Boyd,
Tetrazolium-based assays for cellular vaibility: A critical examination of selected
parameters affecting formazan production, Cancer Research (1991) 51, 2515-2520.
(4) J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna and J.B. Mitchell,
Evaluation of a tetrazolium-based semiautomated colorimetric assay : Assessment of
chemosensitivity testing, Cancer Research (1987) 47, 936-942.
(5) F. Denizot and R. Lang, Rapid colorimetric assay for cell growth and survival,
Journal of Immunological Methods (1986) 89, 271-277.
(6) Letter to the Editor Acid phosphatase: Endpoint for in vitro toxicity tests, In vitro
Cell Dev. Biol. (1991) 27A, 183-184.
(7) D.A. Scudiero, R.H. Shoemaker, K.D. Paull, A. Monks, S. Tierney, T.H. Nofziger,
M.J. Currens, D. Seniff and M.R. Boyd, Evaluation of a soluble tetrazolium/formazan
assay for cell growth and drug sensitivity in culture using human and other tumor cell
lines, Cancer Research (1988) 48, 4827-4833.
(8) R. Fautz, B. Husein and C. Hechenberger, Application of the Neutral Red assay
to monolayer cultures of primary hepatocytes : rapid colorimetric viability
determination for the uncheduled DNA synthesis test (UDS), Mutation Research
(1991) 253, 173-179.
(9) H. Babich, D.W. Rosenberg and E. Borenfreund, In vitro cytotoxicity studies with
the fish hepatoma cell line, PLHC-1 (Poecilliopsis lucida), Ecotoxicology and
environmental safety (1991) 21, 327-336.
(10) C. Legrand, J.M. Bour, C. Jacob, J. Capiaumont, A. Martial, A. Marc, M. Wudtke,

G. Kretzmer, C. Demmangel, D. Duval and J. Hache, Lactate Dehodrogenase
(LDH) activity of the number of dead cells in the medium of cultured eukaryotic cells
as marker, Journal of Biotechnology (1992) 25, 231-243.
(11) T. Decker and M.-L. Lohmann-Matthes, A quick and simple method for the
quantitation of lactate dehydrogenase release in measurements of cellular toxicity
and tumor necrosis factor (TNF) activity, Journal of Immunological Methods (1988)
15, 61-69.
(12) B. Pauwels, A.E.C. Korst, C.M.J. de Pooter, G.G.O. Pattyn, H.A.J. Lambrechts,
M.F.D. Baay, F. Lardon and J. B. Vermorken, Comparison of the sulforhodamine B
assay and the clonogenic assay for in vitro chemoradiation studies, Cancer
Chemother Pharmacol (2003) 51, 221-226.
13) G. Griffon, J.-L. Merlin and C. Marchal, , Comparison of sulforhodamine B,
tetrazolium and clonogenic assays for in vitro radiosensitivity testing in human
ovarian cell lines, Anti-Cancer Drugs (1995) 6, 115-123.
(14) P. Köpf-Maier and B. Kolon, An organoid culture assay (OCA) for determining
the drug sensitivity of human tumors, Int. J. Cancer (1992) 51, 99-107.
(15) P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T.

Warren, H. Bokesch, S. Kenney and M.R. Boyd, New colorimetric cytotoxicity assay
for anticancer-drug screening, Journal of the National Cancer Institute (1990) 82, 13.
Comparison of 4 Cytotoxicity Assays Performed Sequentially on
XENOMETRIX
the Same Cell Sample
by Endotell
Markus Kamber
Xenometrix, Gewerbestr. 25, CH - 4123 Allschwil, Switzerland
Introduction and Summary: Results:

Novel compounds need to be evaluated for unwanted cytotoxic effects early in development. At Figure 1 shows IC50 values of 6 toxicants tested sequentially for LDH, XTT, NR and SRB inhibition. Four
these stages the amount of available test compound is usually limited, yet as much information as different exposure times are shown. The red bar indicates the highest concentration tested; the real IC50
possible on unwanted side-effects should be obtained in order to determine which compounds may be higher. Note that 5-FU was not tested after 2 hrs and overnight exposure due to absence of toxicity
warrant further development. at 24 hrs. p-Phenylenediamine was not tested after 2 hrs exposure.
The evaluation of cytotoxic effects on cell lines or primary cells is a well established and widely
accepted method. Several different cellular targets using various read-outs and different exposure 5-FU Isophorone
protocols are being used. Some assays measure effects on overall survival or proliferation, while
1200 120
others can give more specific insights on toxic mechanisms.
1000 100
Toxicity testing at early stages of development should have a high sensitivity, i.e. effectively toxic
substances should have a very high probability of being identified. Moreover, it should be reasonably 800 80
IC50 (mM)
IC50 (µM)
simple, reproducible, and cost-effective. The PAN I cytotoxicity kit by Xenometrix allows to apply 4
commonly used cytotoxicity assays sequentially on 1 cellular sample. Such an approach has the 600 60
following main advantages: 400 40
- 4 x less test compound consumption than with individual assays 200 20

- Enhanced probability to identify toxic compounds
- Information on possible mechanisms of action 0 0
2h 16 h 24 h 48 h 2h 16 h 24 h 48 h
- Reduced consumption of valuable primary cells
Exposure time Exposure time
- No inter-assay variability due to variations in cell composition, cellular activity, cell
density or other external factors LDHe XTT NR SRB LDHe XTT NR SRB
- Quality controlled reagents and straightforward, highly reproducible protocols.
Here we present a comparative evaluation of

Chlorpromazine Chloroquine
6 cytotoxic compounds in the newly available NEUTRAL RED
- Lysosomal activity
PAN I Cytotoxicity assay kit. This kit combines - Membrane permeability
XTT 250 6000
assays for extracellular Lactate Dehydrogenase LDHe
- Mitochondrial metabolism
- Respiratory toxicity
(LDHe) to measure membrane integrity, the XTT - Membrane
permeability 200
5000
tetrazolium salt to evaluate mitochondrial activity,
4000
Neutral Red (NR) for the evaluation of lysosomal
IC50 (µM)
IC50 (µM)
150
SULFORHODAMINE B
activity, and the Sulforhodamine stain (SRB) for - Total protein
synthesis rate
3000
total protein content. 100
2000
The 4 assays are performed sequentially on the same cell samples in triplicates. This experimental 50
1000
set-up eliminates intra-test variability due to potential variables such as passage number, time-since-
plating, cell density, medium composition (medium age) and others. Differences in IC50 between 0 0
assays reflect therefore more accurately the differential response of cellular functions and 2h 16 h 24 h 48 h 2h 16 h 24 h 48 h
compartments to toxicants because all other sources of variability due to external factors are Exposure time Exposure time
eliminated. Compounds were tested for their cytotoxic activity on L929 mouse fibroblasts after 4
LDHe XTT NR SRB LDHe XTT NR SRB
different exposure times.
The results demonstrate the feasibility of performing 4 different assays sequentially, and time-
dependent examples of differential responses in the assays are presented. The PAN I assay can
p-Phenylenediamine Propranolol
identify compound toxicity with a higher probability than a single assay, and valuable information on
cellular target structures may be obtained. 1600 300
1400
250
Methods: 1200
200
IC50 (µM)
1000
IC50 (µM)
The tests were performed on L929 mouse fibroblasts. Cells were harvested with Trypsin/EDTA and
seeded at 20’000 (2 hr and overnight exposure) or 10’000 (24 and 48 hr exposure) cells per well into 800 150
96-well microtiter plates in DMEM/Ham’s F12 medium supplemented with 10% FCS, Glutamine and 600
100
Penicillin/Streptomycin. 400
50
The cells were allowed to adhere for 6 hrs before addition of test compounds for 2, 16 (overnight), 24, 200
or 48 hrs. 0 0
2h 16 h 24 h 48 h 2h 16 h 24 h 48 h
The following compounds were tested: Exposure time Exposure time
Compound Highest conc. Solvent
LDHe XTT NR SRB LDHe XTT NR SRB
5-Fluorouracil 1 mM DMSO
Isophorone 100 mM TC Medium
Chlorpromazine 500 µM Water Notable findings:
Chloroquine 5 mM Water
p-Phenylenediamine 1,5 mM DMSO 5-FU: Note the strong difference of IC50 between assays at 48 hrs. For toxicants
Propranolol 5 mM DMSO like 5-FU, LDH and XTT are poor indicators of toxicity.
From each compound 8 concentrations, serially diluted in half-log steps were tested in triplicates. Each Chloroquine: In a short time exposure (2 hrs) the Neutral Red assay is very
test was repeated once.
sensitive and suggests a primary toxic effect on the lysosomal compartment. The
Assays for LDH, XTT, NR and SRB were performed sequentially on the same cells according to the other assays show little or now response at 2 hrs.
‘Instructions for Use’ of the InCytotox PAN I kit from Xenometrix.
IC50 values were determined as follows: Chlorpromazine: is a fast acting toxicant with 3 of the 4 assays. LDH toxicity is
A linearized graph was obtained by plotting
measurable only after longer exposure times.
log (%SC/(100%-%SC) vs. log concentration
where Conclusions:
%SC is the OD (measured at an assay-specific wavelength and corrected for the blank value) • The PAN I assay kit allows the sequential measurement of 4
expressed as a percentage of the Solvent Control OD obtained. From such linearized graphs those
data clustering around "0" (= 50% of SC) were used to calculate a linear regression and from this cytotoxicity parameters on 1 cellular sample.
equation the corresponding IC50 was determined. An example is shown below.
• For some compounds (e.g. Propranolol) any of the 4 cytotoxicity
XTT, Chlorpromazine
2 tests yields comparable IC50 values.
2 1.5
1.5
• For other compounds the apparent toxicity depends strongly on the
log (%SC/(100%-%SC)
1
1 assay chosen, and the exposure time.
0.5
0.5
0 0
0.0000 0.5000 1.0000 1.5000 2.0000 2.5000
• Using 4 sequential cytotoxicity tests greatly enhances chances to
-1.0000 0.0000 1.0000 2.0000 3.0000
-0.5
-0.5 detect toxicants which might go unnoticed if only 1 assay were used.
-1
-1.5
-1 y = -2.6579x + 4.7683
R2 = 0.9943
This becomes even more pronounced in experimental set-ups where
log conc. -1.5
only 1 exposure time is used.
In this example the calculated IC50 is 62 µM • The comparison of the kinetic behaviour of the IC50's can provide
All IC50 values obtained by this method were averaged over two independently conducted information on the possible mechanisms of action of test
experiments. compounds.
www.xenometrix.ch
10. CelTox Software
The CelTox software has been developed specifically for the IN CYTOTOX product
range, for the data management, data processing and reporting of the assays. It
consists of three sections. The first one has been designed to contain the raw data of
the assays, or the lab journal. Information on the cell lines used, culture conditions,
test compounds, cytotoxicity assay etc. are stored in the corresponding files. The
results of the measurements of the optical densities are analysed in the analytical
section of the software. The IC50 and/or ID50 values and the linear regressions are
calculated and visualized with the corresponding graphs. The third part consists of a
lexicon where data of cell lines, information on organisms, chemical compounds etc.
can be stored.
The software can be used for all IN CYTOTOX test kits and is available separately.
CelTox Labbook
XENOMETRIX
by Endotell
Electronic Lab Journal
Aim: Traceability of assay conditions
Consists of:
• Database
• Index cards
• Tests
CelTox Index Cards
XENOMETRIX
by Endotell
Consists of:
• Collection of standard data

• Possibility to obtain information from
database
• Possibility to directly add information
to the database
CelTox Analysis
XENOMETRIX
by Endotell
Aim: Automation of cytotoxicity analysis
Analysis in 3 stages:
• Data import or data keyboard

Management of different scheme
plates
• Calculations
• Graphs
CelTox Linearity
XENOMETRIX
by Endotell
• Determination of cell concentration
• Calculation of linear regression
• Linearity range
CelTox Report
XENOMETRIX
by Endotell
Aim:
• Automated analysis report
• Modular report printouts

Cytotoxicity Screening Methods Guide

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XENOMETRIX

2) Objectives of the IN CYTOTOX Assays

b. Combined Test Kits

3) Principle of the Assays

4) IN CYTOTOX Test Kits

5) Advantages of the IN CYTOTOX Test Kits

6) Sensitivities of IN CYTOTOX Assays

7) Aniara Product List (IN CYTOTOX and AMES)

10) CelTox Software

The safety evaluation of compounds such as drugs, cosmetics, food additives,

2. Objectives of the IN CYTOTOX Assays

Alternative methods to animal experimentation allow to limit the use of experimental

The IN CYTOTOX high-throughput test systems allow the measurements of

b. Combined Test Kits

The combined IN CYTOTOX test kits allow to determine up to four metabolic

The IN CYTOTOX assays consist of four steps:

- trypsinization of the cells

Cells are harvested by trypsinisation, counted, diluted and transferred to 96-well

4. IN CYTOTOX Test Kits

One Parameter Kits

XTT and MTT Tetrazolium salt

Multiple Parameter Kits

- Complete range of cytotoxicity markers

- High throughput screening methods

- Single or multiple parameters from one cellular sample

- Specific CelTox software available

- Customized reporting format

- Dedicated customer support by experienced staff

1 5000 0.8 MTT

Cells per well Cells per well

Single Parameter Kits

CVDE Cristal Violet Dye Elution

LDHe Extracellular Lactate Dehydrogenase

MTT Diphenyltetrazolium bromide

PAC Acid Phosphatase

XTT Tetrazolium hydroxide

Multiple Parameter Kits

LDHe - XTT - SRB

XTT - SRB - CVDE

LDHe - GLU - XTT - PAC

LDHe - GLU - XTT - SRB

PAN I : LDHe - XTT - NR - SRB

Celtox Software AICT.Celtox

Plastic Ware (microplates and reservoirs) are available on request:

*The experimental design (number of doses, placement of controls

Ames MPF 98/100 - 10 Samples Kit ATAG-98/100-10

Ames MPF 98/100 - 10 Samples Kit - Rat Liver S9 ATAG-98/100-10-S9

Ames MPF 98/100 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAG-98/100-10-C

Ames MPF 98/100 - 1 Sample Kit ATAG-98/100-1

Ames MPF 98/100 - 1 Sample Kit - Rat Liver S9 ATAG-98/100-1-S9

Ames MPF 98/100 - 1 Sample Kit - Rat Liver S9 - Pos.Control ATAG-98/100-1-C

Ames MPF 98/100/1535/1537 - 10 Samples Kit ATAG-981003537-10

Ames MPF 98/100/1535/1537 - 10 Samples Kit - Rat Liver S9 ATAG-981003537-10-S9

Ames MPF 98/100/1535/1537 - 10 Samples Kit - S9 - Pos.Control ATAG-981003537-10-C

Ames MPF 98/100/1535/1537 - 1 Sample Kit ATAG-981003537-1

Ames MPF 98/100/1535/1537 - 1 Sample Kit - Rat Liver S9 ATAG-981003537-1-S9

Ames MPF 98/100/1535/1537 - 1 Sample Kit - S9 - Pos.Control ATAG-981003537-1-C

Ames MPF PENTA I E.coli Combination - 10 Samples Kit

Ames MPF PENTA I E.coli- Combination - 1 Sample Kit

Ames MPF 98 - 10 Samples Kit ATAG-98-10

Ames MPF 98 - 10 Samples Kit - Rat Liver S9 ATAG-98-10-S9

Ames MPF 98 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAG-98-10-C