Hepatocytes were cultured as mentioned in section 2.1 and 2.2. Once spheroid formation was observed (2–3 days after cell seeding), cells were treated with DMSO or relevant hepatotoxicants (APAP/troglitazone) at different concentrations. 24 hour live cell imaging was performed starting 2 hours after drug addition using IN Cell Analyzer 2000 Imaging System (GE Healthcare Lifesciences, Buckinghamshire, U.K.). Images were acquired every 2 hours. Images acquired at 2 hours, 8 hours, 16 hours and 24 hours were used for further analysis. Individual spheroids were identified by image segmentation using Matlab Image Processing Toolbox and Statistical Toolbox (Mathworks) and the sizes of the spheroids were determined. Density plots were acquired in R (Version 3.1.2) to show the spheroid size distribution and percentage of spheroids within a certain diameter range at different drug concentrations was plotted in Excel.
In cell analyzer 2000 imaging system
Manufactured by GE Healthcare
Sourced in United Kingdom, Sweden
The IN Cell Analyzer 2000 Imaging System is a high-content screening platform designed for automated cellular imaging and analysis. It combines advanced optics, a high-performance camera, and an intuitive software interface to enable quantitative, multiplexed imaging of live or fixed cells.
Automatically generated - may contain errors
3 protocols using in cell analyzer 2000 imaging system
1
High-throughput Spheroid Hepatotoxicity Assay
2
Assessing Acidic Vesicular Organelles with MDC
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The autofluorescent agent MDC (excitation,
360 nm; emission, 535 nm) was utilized to evaluate the abundance of
acidic vesicular organelles (AVOs) in the cells because it can accumulate
in AVOs and exhibit fluoresced bright green dots.22 For qualitative analysis, cells were seeded onto 96-well
plates, treated with indicated concentrations of GA for 24 h and stained
with 50 μM MDC in PBS for 30 min. Cells were washed with PBS
three times and immediately analyzed using In Cell Analyzer 2000 imaging
system (GE Healthcare, Uppsala, Sweden). For qualitative analysis,
cells were seeded onto 24-well plates and treated with a series of
concentrations of the test compounds. Then, cells were stained with
50 μM MDC in PBS for 30 min. After washed with PBS three times,
the mean fluorescence intensities of the cells were determined with
a flow cytometer (Becton Dickinson FACS CantoTM, BD Biosciences, San
Jose, USA).
360 nm; emission, 535 nm) was utilized to evaluate the abundance of
acidic vesicular organelles (AVOs) in the cells because it can accumulate
in AVOs and exhibit fluoresced bright green dots.22 For qualitative analysis, cells were seeded onto 96-well
plates, treated with indicated concentrations of GA for 24 h and stained
with 50 μM MDC in PBS for 30 min. Cells were washed with PBS
three times and immediately analyzed using In Cell Analyzer 2000 imaging
system (GE Healthcare, Uppsala, Sweden). For qualitative analysis,
cells were seeded onto 24-well plates and treated with a series of
concentrations of the test compounds. Then, cells were stained with
50 μM MDC in PBS for 30 min. After washed with PBS three times,
the mean fluorescence intensities of the cells were determined with
a flow cytometer (Becton Dickinson FACS CantoTM, BD Biosciences, San
Jose, USA).
3
Quantifying Cell Proliferation Dynamics
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The cells were seeded at a density of 4 × 104 cells per well and allowed to adhere for 24 h prior to addition of the experimental compounds: CHIR99021 (Cayman Chemical) or the in-house produced proteins APOH, SerpineA6 and STC1 (Novo Nordisk). Cells were stimulated for 24–48 h.
For the 14C-Thymidine incorporation assay, the cells were plated in scintillation plates (CytoStar-T 96-well, Perkin Elmer) and following change to experimental media 14C-Thymidine was supplemented to a final concentration 0.5 μCi/ml. The scintillation signal was measured in a TopCounter NXT HTS instrument (Perkin Elmer).
For the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay the cells were plated in black 96-well culture plates (BD-Falcon) and following change to experimental media the amount of incorporated EdU was analyzed using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies) according to the manufacturer's procedure. The cells were incubated with 0.5 μM EdU for 2 h and subsequently fixed and stained with DAPI. The EdU signal was quantified using the InCell Analyzer 2000 Imaging system (GE Healthcare).
For the 14C-Thymidine incorporation assay, the cells were plated in scintillation plates (CytoStar-T 96-well, Perkin Elmer) and following change to experimental media 14C-Thymidine was supplemented to a final concentration 0.5 μCi/ml. The scintillation signal was measured in a TopCounter NXT HTS instrument (Perkin Elmer).
For the 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay the cells were plated in black 96-well culture plates (BD-Falcon) and following change to experimental media the amount of incorporated EdU was analyzed using the Click-iT EdU Alexa Fluor 488 Imaging Kit (Life Technologies) according to the manufacturer's procedure. The cells were incubated with 0.5 μM EdU for 2 h and subsequently fixed and stained with DAPI. The EdU signal was quantified using the InCell Analyzer 2000 Imaging system (GE Healthcare).
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