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Automated Cytotoxicity and Cell Proliferation Assays
Sponsored,vendor-submitted protocol   Sponsored by Perkinelmer Life Sciences    Published in November 2007 (p.41)

Using the JANUS Cellular Workstation with ATPlite 1step

ATPlite™ 1step is a homogenous, luciferase-based luminescence assay system for the quantitative measurement of proliferation and cytotoxicity in cultured mammalian cells. We describe here a protocol for automating the ATPlite 1step system using the JANUS® Cellular Workstation. ATPlite 1 -step system is used here to measure the cytotoxic dose-response of U937 cells, by exposing cells to Staurosporine, a non-selective tyrosine kinase inhibitor, or Acitnomycin D, which binds DNA and blocks mRNA transcription by RNA polymerase. Results, presented in the full protocol, demonstrate the key performance and pharmacological parameters of the assay with typical Z′ values between 0.82 and 0.85. Hardware:

JANUS Cellular Workstation (Figure 1)


Figure 1 (Click to enlarge)


FlexDrop PLUS EX Precision Reagent Dispenser

Materials:

ATPlite 1step kit (PerkinElmer)

Cell lines

  • HepG2 Cells

  • U937 Cells

Growth medium [MEM (Invitrogen, Inc.), 10% FBS, 2 mM

L-Glutamine, 100 mM sodium pyruvate and 10 mM HEPES]

Staurosporine (Sigma, Inc.)

Actinomycin D (Sigma, Inc.)

384-well CulturPlate™, white (PerkinElmer)

Protocol:

The general ATPLite 1step assay protocol is segmented into 5 steps: Cell Seeding was conducted using the FlexDrop PLUS EX Precision Reagent dispenser in stand-alone mode. All subsequent assay plate processing was fully automated on the JANUS Cellular Workstation. A general outline of each protocol step is provided in the full protocol.

  1. Cell Seeding

  2. Standards/Compound Addition

  3. Assay Incubation

  4. ATPlite 1step Reagent Addition

  5. Assay Plate Reading

Typical Results:

For both compounds, the kinetics of the response was similar with a greater degree of toxicity observed after 48 hours. Actinomycin D demonstrated approximately 10-fold higher potency compared to Staurosporine. Dose-response curves were generated and Z′ values determined. Sample data is shown in Figure 2 with an average Z′ of 0.82 across 10 plates for U937 cells treated with Staurosporine. For complete assay detail and full data sets, please download the complete protocol.


Figure 2 (Click to enlarge)


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