ETC (CRCM) – Marseille, 2018-10-03 · 3 Technologies vs Perturbateurs Luminescence (Lumi) Fluorescence Intensity (FI) Fluorescence Polarization (FP) Alpha Time Resolved Fluorescence

HUMAN HEALTH • ENVIRONMENTAL HEALTH

© 2015 PerkinElmer

ETC (CRCM) – Marseille, 2018-10-03Reynald HURTEAUX – Field Application Specialist

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Background

Les paramètres impliqués dans la qualité d’un cribl age sont principalement:

BiologieTechnologieInstrument

RobotiqueReader

AbsorptionFluorescencePolarisation

AlphaTRF

Luminescence

Type cellulaire, cible

Janus

Ensight

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Technologies vs Perturbateurs

Luminescence (Lumi)

Fluorescence Intensity (FI)

Fluorescence Polarization (FP) Alpha

Time Resolved Fluorescence (TRF)

Absorbance (Abs) Image-based Cytometry

For

tem

ent

Quencher, Autofluorescence, solvants, Temp, viscosité, pH

Quencher, solvants (DMSO) Autofluorescence(quasi nul en Luminescence)

Fai

blem

ent

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Orthogonal Approach (Multi techno) to avoid drawbacks

• Cell Morphology◦ Stainless Brightfield Imaging

• Nucleus Characteristics◦ Fluorescent Imaging Using Nucleus Stain

• Cytoplasmic Enzyme Activity◦ Fluorescent Imaging Using Enzyme Dependant Cytoplasm Stain

• Alpha measurement & Intracellular ATP Levels◦ Luminescence Readout

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Absorbance

Fluorescence Intensity

Luminescence

Time-Resolved

Fluorescence

Alpha Technology

Cellular Imaging

Image-based Cytometry on a Plate Reader

• Uses various detection modes: ◦ fluorescence ◦ brightfield◦ digital phase contrast

• Generates one or more images per well of 96- or 384-well microplates

• Analyzes images automatically during measurement

• Very fast acquisition and analyses

EnSight™ Multimode Plate Reader

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Cytotoxicity

• Cytotoxicity can be caused by different types of agents: ◦ Drugs, pathogens, immune cells and external stress factors

• Cell death includes complex signaling programs that lead to a variety of different phenotypes: ◦ Nuclear fragmentation, cell shrinkage, membrane blebbing or loss of

membrane integrity, leakage of cell content, and swollen nuclei

Cytotoxicity is an ideal application to be analyzed using cell imaging, in addition to conventional cytotoxicity assays [O'Brien, 2014].

+ toxic compound

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Multimodal Cytotoxicity Workflow: Easy and Time-sav ing

Total ERKp-ERK ½p-AKTAutophagy (LC3B / p62)…

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Day 1 (BF measurement)


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Investigate a whole 384-well plate in less than 5 minutes

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Day 1 – Well A1 Day 1 – Well B1

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Day 2 – Well A1 Day 2 – Well B1

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EnSight Brightfield Imaging: Treated vs. Untreated

Well B on Day 1 (Before Treatment)

Well B on Day 2 (Treated with 10% DMSO)

Well A on Day 2 (Untreated)

Well A on Day 1 (Before Treatment)

• HeLa cells were treated with serial dilution of DMSO for 24h

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EnSight Brightfield Imaging: Incorporated confluency analysis

• HeLa cells were treated with serial dilution of DMSO for 24h

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Day 2 (Alpha measurement)


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Add the analyte (5µL in standard protocol)

Add biotinylated anti-analyte antibody

and antibody-conjugated Acceptor beads.

Incubate for 60 minutes.

Excite Donor beads.

Add Streptavidin-coated Donor beads.

Incubate for 30 minutes.

Donor beads release singlet oxygen, activating

Acceptor bead emission.

Molecule up to 200 nm in size can be measured.

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Automated data analysis in separated software

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Day 3 (Fluorescence Imaging)


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Day 3 (Fluorescence Imaging)

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EnSight Fluorescent Imaging: Treated vs. Untreated

• Untreated cells: ◦ Homogenous nucleus and cytoplasm staining

• Treated cells: ◦ Intense stain in small nuclei and only few cells are positive for Calcein AM

• Cells were analyzed using the pre-set Kaleido CytoNuc script

• Hoechst 33342◦ Nucleus stain◦ Stains all cells

• Calcein AM ◦ Enzyme dependant

cytoplasm stain◦ Stains living cells

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Day 3 (Viability by ATP Luminescence kit)


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Luminescence Cytotoxicity Assay: ATPlite 1step kit

• ATPlite 1step kit: ◦ Determines intracellular levels of

ATP◦ Frequently used to detect cell death

• Comparison of ATPlite 1step kit in presence of imaging stain and without

• Plate type: ◦ Black ViewPlate-384

• Plate bottom was covered with a white BackSeal to decrease luminescence crosstalk

• ATPlite 1step kit was added after 2 hours of stain addition (Hoechst and Calcein AM)

Kaleido Luminescence Heat Map

- Stain ← │→ + Stain

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Day 3 (Viability by ATP Luminescence kit)

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Multimodal Cytotoxicity Workflow: Easy and Time-sav ing

Investigate a whole 384-well plate in less than 5 minutes per color

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Advanced data managementExample on BF measurement

• Confluency increases from Day 1 to Day 2 due to cell proliferation• Stainless and non-hazardous brightfield images enable determination

of cytostatic versus cytotoxic effects • In presence of DMSO:

◦ Confluency decreases dose-dependantly due to cell shrinking and loss of cell attachment

◦ Foreground roughness (texture of cell area) increases due to enhanced roundness of dying cells

Brightfield Confluency after Treatment Normalized Brightfield Confluency (After – before treatment)

Foreground Roughness after Treatment

Cytostatic

Cytotoxic

*based on Acapella® High Content Imaging and Analysis Software

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• Dose-response curves are both very similar +/- stain and EC50values are the same

• ATPlite 1step kit is not influenced by the presence of Hoechst and Calcein AM stain

• Imaging and Luminescence measurements can be easily combined in the same well and allows easy orthogonal research

EC50 = 2.41% EC50 = 2.37%

Advanced data managementExample on Intracelllar ATP measurement

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Comparison of EC 50 Values for Multiparametric Cytotoxicity Assays

• Multiparametric approach is able to analyze and distinguish between different levels of cytotoxicity.

Impaired Calcein AM conversion is known to be an early event in apoptotic cells

[Gatti et al., 1998].

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Conclusion

• Gain more data & findings faster out of your sample to confirm your results

• Check cell morphology, cell nucleus & cytoplasm characteristics as well as intracellular ATP levels◦ Get the complete picture of cytotoxicity on different levels

• Watch cell proliferation and compound effects by using stainless brightfield confluency analysis◦ Robust & easy to apply◦ Non-invasive, live cell assay◦ Distinguish between cell static vs. cell toxic effects

• Utilize the power of image segmentation by fluorescent image analysis◦ Distinguishes between nucleus and cytoplasm ◦ Allows for more detailed analysis of different effects of cytotoxicity

• Get confidence in your results by intracellular ATP measurements using Luminescence ◦ Orthogonal: Confirms the well-imaging based results

Orthogonal approachAnalysis of Cytotoxicity

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ETC (CRCM) – Marseille, 2018-10-03 · 3 Technologies vs Perturbateurs Luminescence (Lumi) Fluorescence Intensity (FI) Fluorescence Polarization (FP) Alpha Time Resolved Fluorescence