ss aniara in cytotox tecdoc

• XTT/MTT

• NR Neutral Red

• SRB Sulforhodamine B

• LDH Lactate dehydrogenase

• CVDE Crystal violet dye elution

• Glucose

• PAC Acid Phosphatase

• Combined Kits: 2–4 parametersfrom 1 cellular sample

XENOMETRIX

Cytotoxicity Screening

Xenometrix GmbH

Gewerbestrasse 25

CH-4123 Allschwil

Tel ++41·61·482 14 34

Fax ++41·61·482 20 72

email [email protected]

www.xenometrix.ch

David Wolf

Aniara Logo

David Wolf

Address Banner

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

David Wolf

Address Banner

IN CYTOTOX Cytotoxicity Test Systems

ACID PHOSPHATASE - Lysosomal activity

NEUTRAL RED - Lysosomal activity

- Membrane permeability

SULFORHODAMINE B- Cell proliferation

- Total protein synthesis rate

GLUCOSE - General physiological

cell state - Glucose consumption

MTT / XTT - Mitochondrial metabolism

- Respiratory toxicity LDHe - Membrane permeability

CRYSTAL VIOLET - Cell proliferation

- DNA

XENOMETRIX by Endotell

David Wolf

Address Banner

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.

David Wolf

Address Banner

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

David Wolf

Address Banner

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

David Wolf

Address Banner

David Wolf

Address Banner

6. Sensitivities of In Cytotox Assays

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

LDHe

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 10 20 30 40 50 60 70

Time (min.)

OD

34

0

Medium 1

40000

20000

10000

5000

2500

1250

625

312

156

78

Medium 2

NR

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Mediu

m 1

40000

20000

10000

5000

2500

1250

625

312

156

78

Mediu

m 2

Cells per well

OD

54

0-O

D6

90

CVDE

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

Mediu

m 1

40000

20000

10000

5000

2500

1250

625

312

156

78

Mediu

m 2

Cells per well

OD

54

0-O

D6

90

XTT/MTT

00.20.40.60.8

11.21.41.61.8

2

Mediu

m 1

40000

20000

10000

5000

2500

1250

625

312

156

78

Mediu

m 2

Cells per well

OD

480-O

D690

XTT

MTT

PAC

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.8000

Mediu

m 1

40000

20000

10000

5000

2500

1250

625

312

156

78

Mediu

m 2

Cells per well

OD

40

5-O

D6

90

SRB

0.0000

0.2000

0.4000

0.6000

0.8000

1.0000

1.2000

1.4000

1.6000

1.8000

2.0000

Mediu

m 1

40000

20000

10000

5000

2500

1250

625

312

156

78

Mediu

m 2

Cells per well

OD

54

0-O

D6

90

David Wolf

Address Banner

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

David Wolf

Address Banner

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

David Wolf

Address Banner

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

LDHe - GLU - XTT - PAC

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:

[email protected]

mailto: [email protected]

David Wolf

Address Banner

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

David Wolf

Address Banner

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




David Wolf

Address Banner

Product Art.No.

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





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

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.


rat liver S9


AC10-512

AC10-512-S1

AC10-512-S1-P

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





1 Vial semisolid E.Coli uvrA or pKM101

1 Vial Ampicillin





�

Ames MPF PENTA I E.coli Combination - 1 Sample Kit - Rat Liver S9


rat liver S9

�

Ames MPF PENTA I E.coli Combination - 1 Sample Kit - S9 - Pos.Con.


rat liver S9


AC01-512

AC01-512-S1

AC01-512-S1-P

Ames MPF 98 - 10 Samples Kit


8 Vials Ampicillin



experiments at least 10 compounds in triplicate at 6 concentrations


ATAG-98-10

David

Address Banner

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

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


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

David Wolf

Address Banner

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

David Wolf

Address Banner

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




David Wolf

Address Banner

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




David Wolf

Address Banner

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




David Wolf

Address Banner

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

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

�

Ames MPF 98/100 - 1 Sample Kit

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

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

�

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

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

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

�

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

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

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

�

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

Ames MPF PENTA I E.coli Combination - 10 Samples Kit - Rat Liver S9

Ames MPF PENTA I E.coli Combination - 10 Samples Kit - S9 - Pos.Con.

�

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

Ames MPF PENTA I E.coli Combination - 1 Sample Kit - Rat Liver S9

Ames MPF PENTA I E.coli Combination - 1 Sample Kit - S9 - Pos.Con.

�


Ames MPF 98 - 10 Samples Kit - Rat Liver S9

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

�

Ames MPF 98 - 1 Sample Kit

Ames MPF 98 - 1 Sample Kit - Rat Liver S9

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

�



Ames MPF 100 - 10 Samples Kit - Rat Liver S9 - Pos.Control

�




�




�




ATAM-98/100-10

ATAM-98/100-10-S9

ATAM-98/100-10-C

�

ATAM-98/100-1

ATAM-98/100-1-S9

ATAM-98/100-1-C

�

ATAM-981003537-10

ATAM-981003537-10-S9

ATAM-981003537-10-C

�

ATAM-981003537-1

ATAM-981003537-1-S9

ATAM-981003537-1-C

�

AD10-512

AD10-512-S1

AD10-512-S1-P

�

AD01-512

AD01-512-S1

AD01-512-S1-P

�

ATAM-98-10

ATAM-98-10-S9

ATAM-98-10-C

�

ATAM-98-1

ATAM-98-1-S9

ATAM-98-1-C

�

ATAM-100-10

ATAM-100-10-S9

ATAM-100-10-C

�

ATAM-100-1

ATAM-100-1-S9

ATAM-100-1-C

�

ATAM-1535-10

ATAM-1535-10-S9

ATAM-1535-10-C

�

ATAM-1535-1

ATAM-1535-1-S9

ATAM-1535-1-C

David

Address Banner

Product Art.No.

Ames MPF 1537 - 10 Samples Kit ATAM-1537-10

Ames MPF 1537 - 10 Samples Kit - Rat Liver S9 ATAM-1537-10-S9

Ames MPF 1537 - 10 Samples Kit - Rat Liver S9 - Pos.Control ATAM-1537-10-C

Ames MPF 1537 - 1 Sample Kit ATAM-1537-1

Ames MPF 1537 - 1 Sample Kit - Rat Liver S9 ATAM-1537-1-S9

Ames MPF 1537 - 1 Sample Kit - Rat Liver S9 - Pos.Control ATAM-1537-1-C

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

David Wolf

Address Banner

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: [email protected]

mailto: [email protected]

David Wolf

Address Banner

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




David Wolf

Address Banner

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:


David Wolf

Address Banner


�

IN CYTOTOX

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

0.0000

0.1000

0.2000

0.3000

0.4000

0.5000

0.6000

0.7000

0.8000

0.9000

0 5000 10000 15000 20000 25000 30000 35000 40000

Cell per well

OD

540-O

D690

David Wolf

Address Banner

Version 1.5 01/2007

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.

David Wolf

Address Banner


�

IN CYTOTOX

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

David Wolf

Address Banner

Version 1.5 01/2007



Approximate assay time: - 45 min


reagent reservoirs

GLU Kit content: - Substrate solution - Enzyme solution - Instruction manual - Glucose standard solution

GLU KIT SIZES AND REFERENCE NUMBERS


AKGLU96.400 400

AKGLU96.1200 1200


David Wolf

Address Banner


�

IN CYTOTOX

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

David Wolf

Address Banner

Version 1.5 01/2007

CHNICAL SPECIFICATIONS


Approximate assay time: - 35 min (1h 10 min if read at RT)


reagent reservoirs

LDHE Kit content: - Reconstitution solution - Substrate solution - Activator solution - Instruction manual

LDHE KIT SIZES AND REFERENCE NUMBERS


AKLE96.300 300

AKLE96.1200 1200


David Wolf

Address Banner


�

IN CYTOTOX

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

David Wolf

Address Banner

Version 1.5 01/2007


Absorbance: - 540 nm + 690 nm (reference)

Approximate assay time: - 3 hrs 40 min


reagent reservoirs

NR Kit content: - Wash solution - Labeling solution - Fixing solution - Solubilization solution - Instruction manual

NR KIT SIZES AND REFERENCE NUMBERS


AKRN96.300 300

AKRN96.1200 1200


David Wolf

Address Banner


�

IN CYTOTOX

PAC ACID PHOSPHATASE

APPLICATIONS

Colorimetric assay for the quantification of the lysosomal metabolism of cells in response to pharmaceutical, chemical and environmental compounds, and nutrients.

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.




David Wolf

Address Banner

Version 1.5 01/2007



Approximate assay time: - 2 hr 20 min


reagent reservoirs

PAC Kit content: - Wash solution - Substrate solution - Lysis solution - Stop solution - Instruction manual

PAC KIT SIZES AND REFERENCE NUMBERS


AKPAC96.300 300

AKPAC96.1200 1200


David Wolf

Address Banner


�

IN CYTOTOX

SRB SULFORHODAMINE B

APPLICATIONS

Colorimetric assay for the quantification of the total protein synthesis rate of cells in response to pharmaceutical, chemical and environmental compounds, and nutrients.

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


David Wolf

Address Banner

Version 1.5 01/2007


Absorbance: - 540 nm (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 - Solubilization solution - Instruction manual

SRB KIT SIZES AND REFERENCE NUMBERS


AKSR96.300 300

AKSR96.1200 1200


David Wolf

Address Banner


�

IN CYTOTOX

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

��

��

��

��

David Wolf

Address Banner

Version 1.5 01/2007


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 - Solubilization solution - Instruction manual

- Substrate solution - Buffer solution - 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


David Wolf

Address Banner

Why should I use

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


David Wolf

Address Banner

Comparison XTT versus MTT:

XTT has a higher sensitivity and a higher dynamic range

A)

XTT/MTT, corrected for medium, 15 min

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

40000 20000 10000 5000 2500 1250 625 312 156 78

Cell number

OD

480/5

40 -

OD

690

MTT XTT

B)

XTT/MTT_2, corrected for medium, 2 hr

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

40000 20000 10000 5000 2500 1250 625 312 156 78

Cell number

OD

48

0/5

40

- O

D 6

90

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).

David Wolf

Address Banner


�

IN CYTOTOX

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.








David Wolf

Address Banner

Version 1.5 01/2007


Absorbance: - LDHe: 340 nm - GLU: 540 nm - XTT: 480 (optimum) or 450 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 - Activator solution - Wash solution - Lysis solution - Instruction manual

LDHe - GLU - XTT - PAC KIT SIZES AND REFERENCE NUMBERS


AKLGXP96.300 4 x 300

AKLGXP96.1200 4 x 1200


David Wolf

Address Banner


�

IN CYTOTOX

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

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 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.









David Wolf

Address Banner

Version 1.5 01/2007


Absorbance: - LDHe: 340 nm - GLU: 540 nm - XTT: 480 (optimum) or 450 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 - Substrate solutions - Activator solution - Labeling solution - Wash solution - Solubilization solution - Rinsing solution - Fixing solution - Instruction manual

LDHe GLU XTT SRB KIT SIZES AND REFERENCE NUMBERS


AKLGXS96.300 4 x 300

AKLGXS96.1200 4 x 1200


David Wolf

Address Banner


�

IN CYTOTOX

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

This kit allows to measure sequentially four cytotoxicity parameters in one single cell 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.









David Wolf

Address Banner

Version 1.5 01/2007


Absorbance: - LDHe: 340 nm - XTT: 480 (optimum) or 450 nm - 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 - Substrate solutions - Activator solution - Labeling solution - Wash solutions - Solubilization solutions - Rinsing solution - Fixing solutions - Instruction manual

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


David Wolf

Address Banner

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 celllines, 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.

David Wolf

Address Banner

(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.

David Wolf

Address Banner

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.

David Wolf

Address Banner


Comparison of 4 Cytotoxicity Assays Performed Sequentially on the Same Cell SampleMarkus Kamber Xenometrix, Gewerbestr. 25, CH - 4123 Allschwil, Switzerland

www.xenometrix.ch

Introduction and Summary:

Novel compounds need to be evaluated for unwanted cytotoxic effects early in development. At these stages the amount of available test compound is usually limited, yet as much information as

possible on unwanted side-effects should be obtained in order to determine which compoundswarrant further development.

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

protocols are being used. Some assays measure effects on overall survival or proliferation, while others can give more specific insights on toxic mechanisms.

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

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 following main advantages:

- 4 x less test compound consumption than with individual assays- Enhanced probability to identify toxic compounds

- Information on possible mechanisms of action- Reduced consumption of valuable primary cells- No inter-assay variability due to variations in cell composition, cellular activity, cell

density or other external factors- Quality controlled reagents and straightforward, highly reproducible protocols.

Here we present a comparative evaluation of 6 cytotoxic compounds in the newly available PAN I Cytotoxicity assay kit. This kit combines

assays for extracellular Lactate Dehydrogenase (LDHe) to measure membrane integrity, the XTTtetrazolium salt to evaluate mitochondrial activity, Neutral Red (NR) for the evaluation of lysosomal

activity, and the Sulforhodamine stain (SRB) for total protein content.

The 4 assays are performed sequentially on the same cell samples in triplicates. This experimental 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 assays reflect therefore more accurately the differential response of cellular functions and compartments to toxicants because all other sources of variability due to external factors are

eliminated. Compounds were tested for their cytotoxic activity on L929 mouse fibroblasts after 4 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

identify compound toxicity with a higher probability than a single assay, and valuable information on cellular target structures may be obtained.

Methods:

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 96-well microtiter plates in DMEM/Ham’s F12 medium supplemented with 10% FCS, Glutamine and

Penicillin/Streptomycin.

The cells were allowed to adhere for 6 hrs before addition of test compounds for 2, 16 (overnight), 24, or 48 hrs.

The following compounds were tested:

From each compound 8 concentrations, serially diluted in half-log steps were tested in triplicates. Each test was repeated once.

Assays for LDH, XTT, NR and SRB were performed sequentially on the same cells according to the

‘Instructions for Use’ of the InCytotox PAN I kit from Xenometrix.

IC50 values were determined as follows:

A linearized graph was obtained by plotting

log (%SC/(100%-%SC) vs. log concentration

where

%SC is the OD (measured at an assay-specific wavelength and corrected for the blank value)

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 equation the corresponding IC50 was determined. An example is shown below.

In this example the calculated IC50 is 62 µM

All IC50 values obtained by this method were averaged over two independently conducted experiments.

Compound Highest conc. Solvent

5-Fluorouracil 1 mM DMSO

Isophorone 100 mM TC Medium

Chlorpromazine 500 µM Water

Chloroquine 5 mM Water

p-Phenylenediamine 1,5 mM DMSO

Propranolol 5 mM DMSO

y = -2.6579x + 4.7683

R2 = 0.9943

-1.5

-1

-0.5

0

0.5

1

1.5

2

0.0000 0.5000 1.0000 1.5000 2.0000 2.5000

XTT, Chlorpromazine

-1.5

-1

-0.5

0

0.5

1

1.5

2

-1.0000 0.0000 1.0000 2.0000 3.0000

log conc.

log

(%

SC

/(100%

-%S

C)

Conclusions:

• The PAN I assay kit allows the sequential measurement of 4 cytotoxicity parameters on 1 cellular sample.

• For some compounds (e.g. Propranolol) any of the 4 cytotoxicity tests yields comparable IC50 values.

• For other compounds the apparent toxicity depends strongly on the assay chosen, and the exposure time.

• Using 4 sequential cytotoxicity tests greatly enhances chances to detect toxicants which might go unnoticed if only 1 assay were used. This becomes even more pronounced in experimental set-ups where

only 1 exposure time is used.

• The comparison of the kinetic behaviour of the IC50's can provide information on the possible mechanisms of action of test compounds.

Results:

Figure 1 shows IC50 values of 6 toxicants tested sequentially for LDH, XTT, NR and SRB inhibition. Four different exposure times are shown. The red bar indicates the highest concentration tested; the real IC50

may be higher. Note that 5-FU was not tested after 2 hrs and overnight exposure due to absence of toxicity at 24 hrs. p-Phenylenediamine was not tested after 2 hrs exposure.

Notable findings:

5-FU: Note the strong difference of IC50 between assays at 48 hrs. For toxicants

like 5-FU, LDH and XTT are poor indicators of toxicity.

Chloroquine: In a short time exposure (2 hrs) the Neutral Red assay is very

sensitive and suggests a primary toxic effect on the lysosomal compartment. The

other assays show little or now response at 2 hrs.

Chlorpromazine: is a fast acting toxicant with 3 of the 4 assays. LDH toxicity is

measurable only after longer exposure times.

NEUTRAL RED - Lysosomal activity

- Membrane permeability

XTT - Mitochondrial metabolism

- Respiratory toxicity LDHe - Membrane permeability

SULFORHODAMINE B - Total protein synthesis rate

5-FU

0

200

400

600

800

1000

1200

2 h 16 h 24 h 48 h

Exposure time

IC5

0 (

µM

)

LDHe XTT NR SRB

Isophorone

0

20

40

60

80

100

120

2 h 16 h 24 h 48 h

Exposure time

IC5

0 (

mM

)

LDHe XTT NR SRB

Chlorpromazine

0

50

100

150

200

250

2 h 16 h 24 h 48 h

Exposure time

IC5

0 (

µM

)

LDHe XTT NR SRB

Chloroquine

0

1000

2000

3000

4000

5000

6000

2 h 16 h 24 h 48 h

Exposure time

IC5

0 (

µM

)

LDHe XTT NR SRB

p-Phenylenediamine

0

200

400

600

800

1000

1200

1400

1600

2 h 16 h 24 h 48 h

Exposure time

IC5

0 (

µM

)

LDHe XTT NR SRB

Propranolol

0

50

100

150

200

250

300

2 h 16 h 24 h 48 h

Exposure time

IC5

0 (

µM

)

LDHe XTT NR SRB

David Wolf

Address Banner

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.

David Wolf

Address Banner


CelTox Labbook

Electronic Lab Journal

Aim: Traceability of assay conditions

Consists of:

• Database

• Index cards

• Tests

David Wolf

Address Banner


CelTox Index Cards

Consists of:

• Collection of standard data

• Possibility to obtain information from database

• Possibility to directly add information to the database

David Wolf

Address Banner


CelTox Analysis

Aim: Automation of cytotoxicity analysis Analysis in 3 stages:

• Data import or data keyboard Management of different scheme plates

• Calculations

• Graphs

David Wolf

Address Banner


CelTox Linearity

• Determination of cell concentration

• Calculation of linear regression

• Linearity range

David Wolf

Address Banner


CelTox Report

Aim:

• Automated analysis report

• Modular report printouts

David Wolf

Address Banner

ss aniara in cytotox tecdoc

Documents