Limits...
In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening.

Niles AL, Moravec RA, Riss TL - Curr Chem Genomics (2009)

Bottom Line: In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities.The early and routine implementation of this testing is testament to its prognostic importance for humans.We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method.

View Article: PubMed Central - PubMed

Affiliation: Research Department, Promega Corporation, 2800 Woods Hollow Road, Madison, WI, USA. andrew.niles@promega.com

ABSTRACT
In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats.

No MeSH data available.


Multi-Parametric Viability Assays Paired with Caspase Assays. Jurkat cells were plated in 384 well plates and treated with serial dosages of etoposide for 6hr. MultiTox-Fluor™ or MultiTox-Glo Reagents (Promega Corporation) were added to the plates and viability and cytotoxicity values measured. Apo-ONE® or Caspase-Glo® Reagents (Promega Corporation) were then added and caspase activation measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2802765&req=5

Figure 7: Multi-Parametric Viability Assays Paired with Caspase Assays. Jurkat cells were plated in 384 well plates and treated with serial dosages of etoposide for 6hr. MultiTox-Fluor™ or MultiTox-Glo Reagents (Promega Corporation) were added to the plates and viability and cytotoxicity values measured. Apo-ONE® or Caspase-Glo® Reagents (Promega Corporation) were then added and caspase activation measured.

Mentions: Triplexes created by uniting the fluorescent viability and cytotoxicity multiplex with luminescent chemistries are advantageous for several reasons. First, it is possible to conduct immediate confirmation testing of apparent cytotoxic compounds in the same well by leveraging the statistical power of orthogonal detection of viability (viable cell aminopeptidase, dead cell protease and ATP biomarkers). The economy of this consolidated testing is noteworthy in terms of compound usage, culture set up, and operator man-hours. Second, the mechanism of cell death can often be elucidated when combined with caspase detection reagents, allowing for partial characterization of a compound’s potential. It should be noted however, that caspase activity can be transient and immeasurable in long-term experiments due to the kinetics of apoptosis and caspase biomarker decay. Therefore, caspase-negative data sets should be rescreened at multiple compound exposure periods. This type of characterization of pathway dependent cytotoxicity is useful for continued annotation of a library, because compounds causing necrotic profiles at micromolar concentrations are rarely therapeutically useful and may offer unacceptable cytotoxic risk. And third, viability and cytotoxicity can be used to normalize luminescent data from other assays (Fig. 7). Single parameter genetic reporter responses are susceptible to experimental variation due to differential transfection efficiencies, cell number delivered to the well (clumping) or to cytotoxicity caused by the test compound [55]. Viability and cytotoxicity data can therefore help reconcile aberrant data points or explain decreases in genetic reporter signals due to cell death.


In vitro viability and cytotoxicity testing and same-well multi-parametric combinations for high throughput screening.

Niles AL, Moravec RA, Riss TL - Curr Chem Genomics (2009)

Multi-Parametric Viability Assays Paired with Caspase Assays. Jurkat cells were plated in 384 well plates and treated with serial dosages of etoposide for 6hr. MultiTox-Fluor™ or MultiTox-Glo Reagents (Promega Corporation) were added to the plates and viability and cytotoxicity values measured. Apo-ONE® or Caspase-Glo® Reagents (Promega Corporation) were then added and caspase activation measured.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802765&req=5

Figure 7: Multi-Parametric Viability Assays Paired with Caspase Assays. Jurkat cells were plated in 384 well plates and treated with serial dosages of etoposide for 6hr. MultiTox-Fluor™ or MultiTox-Glo Reagents (Promega Corporation) were added to the plates and viability and cytotoxicity values measured. Apo-ONE® or Caspase-Glo® Reagents (Promega Corporation) were then added and caspase activation measured.
Mentions: Triplexes created by uniting the fluorescent viability and cytotoxicity multiplex with luminescent chemistries are advantageous for several reasons. First, it is possible to conduct immediate confirmation testing of apparent cytotoxic compounds in the same well by leveraging the statistical power of orthogonal detection of viability (viable cell aminopeptidase, dead cell protease and ATP biomarkers). The economy of this consolidated testing is noteworthy in terms of compound usage, culture set up, and operator man-hours. Second, the mechanism of cell death can often be elucidated when combined with caspase detection reagents, allowing for partial characterization of a compound’s potential. It should be noted however, that caspase activity can be transient and immeasurable in long-term experiments due to the kinetics of apoptosis and caspase biomarker decay. Therefore, caspase-negative data sets should be rescreened at multiple compound exposure periods. This type of characterization of pathway dependent cytotoxicity is useful for continued annotation of a library, because compounds causing necrotic profiles at micromolar concentrations are rarely therapeutically useful and may offer unacceptable cytotoxic risk. And third, viability and cytotoxicity can be used to normalize luminescent data from other assays (Fig. 7). Single parameter genetic reporter responses are susceptible to experimental variation due to differential transfection efficiencies, cell number delivered to the well (clumping) or to cytotoxicity caused by the test compound [55]. Viability and cytotoxicity data can therefore help reconcile aberrant data points or explain decreases in genetic reporter signals due to cell death.

Bottom Line: In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities.The early and routine implementation of this testing is testament to its prognostic importance for humans.We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method.

View Article: PubMed Central - PubMed

Affiliation: Research Department, Promega Corporation, 2800 Woods Hollow Road, Madison, WI, USA. andrew.niles@promega.com

ABSTRACT
In vitro cytotoxicity testing has become an integral aspect of drug discovery because it is a convenient, costeffective, and predictive means of characterizing the toxic potential of new chemical entities. The early and routine implementation of this testing is testament to its prognostic importance for humans. Although a plethora of assay chemistries and methods exist for 96-well formats, few are practical and sufficiently sensitive enough for application in high throughput screening (HTS). Here we briefly describe a handful of the currently most robust and validated HTS assays for accurate and efficient assessment of cytotoxic risk. We also provide guidance for successful HTS implementation and discuss unique merits and detractions inherent in each method. Lastly, we discuss the advantages of combining specific HTS compatible assays into multi-parametric, same-well formats.

No MeSH data available.