Veritas™ Microplate Luminometer Method for

aCella™ -TOX

1. INTRODUCTION

The Turner BioSystems' Veritas™ Microplate Luminometer in combination with Cell Technology's aCella™-TOX kit provides a sensitive assay for detecting cell cytotoxicity. Cell Technology's assay for cell cytotoxicity quantitatively measures the release of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from mammalian cell lines or bacterial cells.(^1,2,3) The aCella-TOX assay can detect cell cytotoxicity in as low as 5 cells/ 100 µL (assay limited).

GAPDH catalyzes the oxidative phosphorylation of glyceraldehyde-3-phosphate (GAP) to 1,3-Bisphosphoglycerate (1,3-BPG) within the glycolytic pathway. Subsequently, 1,3-BPG is dephosphorylated by the enzyme phosphoglycerate kinase (PGK) to produce ATP, which is then detected via the luciferase/luciferin bioluminescence methodology.

The Veritas Microplate Luminometer employs a unique system for light detection to maximize the sensitivity and range of the aCella-TOX assay kit. All tests were conducted using the aCella-TOX assay kit (Cell Technology, Inc. Catalog #CLATOX 100-3, #CLATOX 100-4).

Figure 1: Jurkat cells were plated at various cell concentrations per well. NP-40 cytotoxic agent was added to each well. The aCella-TOX kit was used to detect G3PDH enzyme release. Data points show average RLU in triplicate.

2. MATERIALS REQUIRED

· Veritas Microplate Luminometer
(P/N 9100-000)
· 96-well plates, opaque white
(E&K Scientific EK-25075)
· Cell Technology aCella-TOX assay kit

3. EXPERIMENT PROTOCOL

3.1 Reagent Preparation

Vial A: Enzyme Assay Reagent (4X)
Bottle A: Enzyme Assay Diluent A
Vial B: Detection Reagent (50X). Contains Luciferase/Luciferin.
Bottle B: Detection Assay Diluent B (5.5X)
Vial C: Glyceradehyde 3-Phosphate (G3P)
Vial D: 5X Lytic agent

3.1.1 Prior to use, thaw vial A (4X concentrate) and keep on ice. To make a 2X working stock, dilute the 4X concentrate 1:2 with Enzyme Assay Diluent A: Bottle A.

3.1.2 Aliquot and freeze the rest of Vial A at less than -70°C.

3.1.3 Next, quickly thaw Vial C. Add 3.5 µL of Vial C to each mL of 2X Enzyme Assay Reagent. Make enough of Enzyme Assay Reagent for a full day of use. You may store the rest of the 2X Enzyme Assay Reagent mixture for 2 weeks at less than -70°C without significant loss of activity.

3.1.4 Thaw Bottle B (5.5X concentrate). Dilute the contents 1:5.5 with reagent grade deionized water prior to use.

3.1.5 Aliquot leftover material and store between -20°C to -70°C.

3.1.6 Thaw Vial B (Detection reagent, 50X concentrate) prior to use and keep on ice.

3.1.7 Aliquot and freeze the rest at less than -70°C.

3.1.8 Just before adding the detection reagent to your sample, dilute it 1:50 with Detection Assay Diluent B (from above).

3.1.9 Add 50 µL to each sample.

NOTE: Keep contents protected from direct light.

3.1.10 Dilute Vial D 1:5 in deionized water prior to use. Diluted material may be stored at room temperature.

NOTE: Using other lytic reagents may interfere with assay signal. TritonX 100 will severely reduce the assay signal.

NOTE: Avoid repeated freeze thaw cycles of all components.

3.2 Instrument Setup

3.2.1 Double click on the Veritas icon to start the software.

3.2.2 Click on "Create New Protocol" from the "Welcome to Veritas" dialog box.

3.2.3 Select "0 injectors" when prompted on the Create New Protocol Wizard.

3.2.4 Modify the delay before starting first run, number of runs, or select a delay time between runs. For "Integration time" select 1 second.

3.2.5 Next, select the wells to be read.
You may "Save Protocol As" for future use, or select "Finish" to run the protocol without saving.

3.2.6 Enter your information into the "Experiment," "Operator," "Plate No." and "Notes" fields in the "Main Dialog Box".

3.3 Sample Analysis-Cytotoxicity

3.3.1 Controls

A. Negative Control
1. Plate cell culture media alone, in triplicate, to measure background signal.

2. In addition, you may plate untreated cells or cells treated with the solvent/vehicle used to dissolve test compound.

B. Positive Control

1. Treat cells with the lytic agent to measure total GAPDH released. Add 5-10 µL of 0.2% NP-40 per 100 µL of sample.

NOTE: Some cell lines may require higher concentrations of NP40 for lysis. It is recommended to use the lowest lysing concentration possible.

3.3.2 Plate, in triplicate, 500-10,000 cells per well in a total volume of 100 µL. Cells may be plated in reduced serum or 10% serum media. Although, when using 10% serum supplemented media it may be necessary to plate between 10,000-20,000 cells per well.

TECHNICAL NOTE 1: Before starting experiments, it is important to determine the linear response range of aCella-TOX within your particular cell line, since GAPDH expression will vary between cell lines.

· Titrate cells in assay media (Cell Technology recommends 1,000-20,000 cells/well). Then add lytic agent to each well and incubate for the length of your assay. Next, add enzyme assay reagent and detection reagent and measure luminescence. For further experiments, use the cell concentration that falls in the linear range of the assay.

3.3.3 Add cytotoxic compounds to your sample.

3.3.4 After incubation according to your experimental protocol:

A. Add 5-10 µL of lytic agent to your positive control sample.

B. Then add 100 µL of 2X Eynzyme Assay Reagent to each well, including positive control.

3.3.5 Immediately add 50 µL of diluted (1:50) Detection Reagent (Vial B) to each well. It may be necessary to incubate the sample for 2-5 minutes in order to achieve maximum signal.

TECHNICAL NOTE 2: Prolonged incubation can cause your assay to fall out of linear range. The enzyme reagents are constantly producing ATP until one of the components becomes exhausted. Take several time point readings from 5-25 minutes to get optimized linear readout.

3.3.6 Insert sample plate into the Veritas Microplate Luminometer and click "Start" to begin assay. RLU values measured by the Veritas will appear in the Excel spreadsheet after all the selected wells in each row have been read. If you encounter an error message, refer to the troubleshooting guide for more information.

NOTE: Opening another Excel Spreadsheet while the Veritas reads your sample plate is strongly discouraged.

3.3.7 Once the measurements are complete you can access Excel to analyze your data.

3.3.8 Remove sample plate after measurements.

3.3.9 Calculation of Results

A. % Cytotoxicity = 100 X (experimental sample - negative control) / (maximum GAPDH release - negative control)

3.4 Sample Analysis-Cell Mediated / Antibody Mediated Cytotoxicity

3.4.1 Controls

A. Negative control

1. Measure, in triplicate, spontaneous release of GAPDH from target cells alone and effector cells alone. Before starting, see Technical Note #1.

B. Positive control

1. Plate, in triplicate, target cells alone. Treat cells with the lytic agent (vial D: 5-10 µL per well) to measure maximum GAPDH released.

3.4.2 Plate between 500-10,000 target cells per well.

NOTE: For antibody-mediated cytotoxicity, target cells should be incubated with the antibody of interest and washed to remove excess antibody, prior to plating. The presence of excess antibody will not interfere with the aCella-TOX signal.

3.4.3 Add effector cells at various effector to target cell ratios

3.4.4 After incubation according to your experimental protocol, Add 5-10 mL of lytic reagent to your target cell positive control. Wait 5 minutes.

3.4.5 Next, add 100 mL of 2X Enzyme Assay Reagent (vial A) to each well.

3.4.6 Immediately add 50 mL of diluted (1:50) Detection Reagent (vial B) to each well. It may be necessary to incubate the sample for 2-5 minutes in order to achieve maximum signal. See Technical Note #2.

3.4.7 Insert sample plate into the Veritas Microplate Luminometer and click "Start" to begin assay. RLU values measured by the Veritas will appear in the Excel spreadsheet after all the selected wells in each row have been read. If you encounter an error message, refer to the troubleshooting guide for more information.

NOTE: Opening another Excel Spreadsheet while the Veritas reads your sample plate is strongly discouraged.

3.4.8 Once the measurements are complete you can access Excel to analyze your data.

3.4.9 Remove sample plate

3.4.10 Calculation of Results

A. %Cytotoxicity Target:Effector Ratio 1:1 = 100 X [(B1 Average - A average) - C1 Average] / (E-D)

· A = spontaneous GAPDH release from target cells alone
· B = GAPDH release from target cells by effector cells (Target:Effector)
· C = spontaneous GAPDH release from effector cells alone, at various effector cell ratios
· D = media alone, background
· E = maximum GAPDH release from target cells

4. REFERENCES

1. Methods and compositions for coupled luminescent assays. United States Patent 6,811,990 Corey and Kinders, issued November 2, 2004.
2. Corey, M. J. and Kinders, R. J. (2005) "Coupled
Luminescent Methods in Drug Discovery: 3-Min Assays for Cytotoxicity and Phosphatase Activity" Drug Discovery Handbook, Ed. Shayne Cox Gad, published by John Wiley & Sons, Inc., pp. 689-731
3. Corey, M.J., et al., "A Very Sensitive Coupled Luminescent Assay for Cytoxicity and Complement-Mediated Lysis," Journal of Immunological Methods 207:43-51, 1997.

5. ABOUT CELL TECHNOLOGY, INC

aCella™ - TOX is a trademark of Cell Technology, Inc. Orders for Cell Technology's products may be placed by:

Phone: (650) 960-2170
Fax: (650) 960-0367
Email: techsupport@celltechnology.com
www.celltechnology.com

Mailing Address: Cell Technology Inc
950 Rengstorff Ave
Suite D
Mountain View, CA 94043
USA

* Special thanks to Cell Technology for the use of their data.

6. ABOUT TURNERBIOSYSTEMS, INC.

Veritas is a trademark of Turner BioSystems. Orders for Turner BioSystems' products may be placed by:

Phone: (408) 636-2400
Toll Free: (888) 636-2401 or
Fax: (408) 737-7919

Contact us via our contact form
www.turnerbiosystems.com

Mailing Address:
Turner BioSystems, Inc.
645 N. Mary Avenue
Sunnyvale, CA 94085

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