Cell Based Assays

Description

This application is an assessment of the High Content Screening solution applied to cytotoxicity assays using living cells. Data generated by label-free DHM®imaging are validated with fluorescence-based methods.

Label-free DHM® imaging is an advantageous read-out for phenotypic (morphology based) screening of living cells for various High Throughput Screening (HTS) and High Content Screening applications. In the present application note, we show that DHM® easily delivers cell viability data and results comparable to fluorescence-based methods in a faster and more effective way.

An important range of early drug discovery applications can be addressed by the DHM® technology, in particular for cell death assays of great importance for toxicological profiling of bioactive compounds, for the search of cytotoxic agents in cancer research or cytoprotective compounds in the context of various therapeutic applications.

 

Material and methods

  • Cell HELA, H9c2
  • Selected toxic compounds for DHM® end point and time lapse measurements and comparisons with fluorescence-based methods: HgCl2, Chloroquine, Gambogic acid, Doxorubicin, Cycloheximide
  • DHM® solution: HCS system equipped with an optional fluorescence module
  • Automatized XY stage and environmental chamber for temperature and CO2 controlled environment (Chamlide WP incubator system, LCI). Use of commercially available 96 and 384-well plates (BD-Falcon imaging plates)
  • Data acquisition. Determination of cell viability with DHM®: label-free, no labeling step required. Recording of OPD signals and cell confluence. Determination of cell viability using fluorescence: PrestoBlue for total well signal measurements using Safire2 (Tecan) multimode reader; Hoechst 33342 and Propidium iodide for cell labeling imaged with a BD Pathway 435 automated microscope.
  • Data analysis. Image segmentation, quantification and analysis using the open-source software CellProfiler (www.cellprofiler.org) for both DHM® and fluorescence microscopy measurements. Quantitative cell population analyses can be performed from DHM® images by measuring confluence and signal recorded as Optical path OPD for both end point and time-lapse measurements.
  • Screening data clustering: Compounds were clustered according to their phenotype: confluence vs. OPD signal for a given cell line and OPD signal for a given cell line against OPD for a second cell line
Fig 1 : Control well
Fig 2 : HeLa cells treated with Doxorubicin

Results

A Z’-factor close to 0.9 was obtained for the cell viability assay using HeLa and H9c2 cells in 96 or 384 well plates, validating the robustness of DHM® assay for phenotypic screening. IC50 curves generated for selected compounds are in excellent agreement between DHM and standard fluorescence-based methods [1]. Clustering of compounds in OPD vs. confluence curves (indicating cytotoxicity or proliferation, respectively) for the whole screen of 1200 drugs facilitate a preliminary classification of compounds according to the cell fate and allowing a fast selection of hits for further analyses such as dose response and time lapse measurements (Fig.3). Running comparative screens using different cell types allows selecting profiled compounds according to the phenotypes generated as illustrated in a proof of principle exercise of toxicological predictive value. Moreover, the highly informative aspect of the analyzed data provide insights about the cellular phenotypes generated and possible indications about the mechanism of action of the drugs for a given cell type. This contributes to the annotation of compounds for an appropriate selection or prioritization of screening hits.

Finally, the convenient utilisation of DHM® for time-lapse experiments during several days for various experimental conditions represents an important advantage in terms of real time monitoring of cellular events provoked by the action of interfering compounds. [2].

In summary, label-free quantitative DHM® imaging is an advantageous technique delivering both HTS screening and HCS data by the combination of easily implemented, fast and cost-effective methodological approaches.

Cell Based Assay Time-lapse

Fig 3 : Timelapse recording of dose-response for Doxorubicin in HeLa cells. Average OPD is the raw DHM signal obtained over the whole cell population whereas “round” phenotype (corresponding to cytotoxicity) is the time-consuming classification achieved on single cells using Cellprofiler (by using the same data set).

 

Publications

[1] Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy
[2] Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

 

Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy

Label-Free Cytotoxicity Screening Assay by Digital Holographic Microscopy

We introduce a label-free technology based on digital holographic microscopy (DHM) with applicability for screening by imaging, and we demonstrate its capability for cytotoxicity assessment using mammalian living cells. For this first high content screening …

Source: www.ncbi.nlm.nih.gov/pmc/articles/PMC3593696/

Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive …

Source: www.ncbi.nlm.nih.gov/pmc/articles/PMC3894694/

Description

Cell migration is central to a variety of functions such as wound healing, cell differentiation, embryonic development and the metastasis of tumours. A better understanding of the mechanism by which cells migrate may lead to the development of novel therapeutic strategies in particular for cancer research where rated metastasis and tumour invasion appear as the main applications of cell migration assays.

In the present screening application, the label-free DHM® imaging provide an informative and fast detection method of active compounds inhibiting cell proliferation/migration using both time-lapse and end point measurements. Moreover, active compounds can be categorized according to their potency through the generation of dose-responses but also according to the phenotype generated. In addition cytotoxic measurement is obtained without further post-processing or analysis using the average OPD information.

In conclusion, our methodological approach allows easy and cost-effective characterization of hits for their ability to perturb cell migration and simultaneously to gather valuable information related to cell phenotypic changes induced by the effect of the chemical compounds.

Material and methods

  • Cell: HeLa cells were plated in OrisTM-Pro 96-well plates (Platypus Technologies). Briefly, silicon-based stoppers provide a temporary physical barrier preventing cells adherence to the center of the well generating an annular monolayer of cells with a central cell-free area (exclusion zone) into which cell movement can occur
  • Toxic compounds used for cell treatments: Cells were treated for 40 h with increasing concentrations of Cytochalasin D (n=3). This compound is a cell permeable potent inhibitor of the polymerization and the elongation of actin resulting in cell cycle arrest (at the G1-S transition) through the activation of p53-dependent pathways.
  • DHM® solution: HCS system. Image acquisition and analysis using a 10x/0.22 NA objective, 25 images per well were recorded at the speed of about 2 images/second leading to a total time of 20 minutes for acquiring 2400 images of an entire 96 well plate. Time-points were acquired each hour for 40 h. Confluency, the readout for migration, was measured by simple thresholding of the images and the average OPD, related to cytotoxicity was measured using the average DHM® signal.

Results

An entire plate has been imaged in time-lapse mode for the different assay conditions. The EC50 value calculated from the dose response curve generated at the end point 40h for the Cytochalasin D is in agreement with previously reported data. High content temporal and spatial information both have been easily generated with our label-free DHM® imaging approach. Chemical compounds can be evaluated and quantified for their ability to prevent cell migration. Moreover, during the time-lapse measurements, phenotypic changes of cells can be recorded giving additional valuable information of the compounds action during time at a given concentration.

Our method is suitable for large scale screening at single compound concentration and focused high content analysis of selected molecules during hits-validation or hits-to-leads process.

Migration Assay t=40h