Plates were imaged using an INCell 1000 automated microscope (GE Healthcare). Images were taken in nine randomly distributed fields in each well of a 96-well plate. The position of each field remained constant in between wells. Image stacks were analysed by an in-house written protocol using the INCell Developer software (GE Healthcare) (Uusimaa et al., 2014 (link); Diot et al., 2015 (link)).
In cell developer software
Manufactured by GE Healthcare
The IN Cell Developer software is a high-content screening and analysis tool designed for cellular imaging applications. It provides automated image acquisition, processing, and data analysis capabilities to support a wide range of cell-based assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
In cell analyzer 2000, by GE Healthcare (5 mentions)
384 well clear black plates, by Greiner (2 mentions)
Hoescht 33342, by Thermo Fisher Scientific (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
In cell analyser 1000, by GE Healthcare (1 mentions)
Anti acetylated tubulin antibody, by Merck Group (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Merck Group (1 mentions)
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
In cell 2200 automated microscope, by GE Healthcare (1 mentions)
Alexa fluor 546, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Anti lama2, by Enzo Life Sciences (1 mentions)
Vectashield media, by Vector Laboratories (1 mentions)
Sp5 microscope, by Leica (1 mentions)
Alexa fluor tagged secondary antibodies, by Thermo Fisher Scientific (1 mentions)
Mouse anti vimentin, by Merck Group (1 mentions)
Z fix, by Anatech (1 mentions)
Ab4074, by Abcam (1 mentions)
Donkey anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
18 protocols using in cell developer software
1
High-Throughput Automated Microscopy Imaging
2
Automated Imaging and Analysis Protocol
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Images were taken on an IN Cell 1000 Analyser (GE Healthcare) automated system. Images were processed and analyzed using GE IN Cell Developer software (Version 1.9.1).
3
High-throughput Screening of Ciliary Dynamics
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CFPAC-1 cells were seeded in 96-well microplates at a density of 10, 000 cells/well containing 200 μl culture medium supplemented with 10% FBS and incubated for 48 hours in a humidified incubator (37°C and 5% CO2), after which medium was refreshed with 200 μl of RPMI-1640 medium containing 2% FBS. The Pharmakon 1600 chemical library consisting of 1600 clinically evaluated compounds and marketed drugs was procured from MicroSource Discovery Systems Inc. (U.S.A). The compounds were dissolved as 10 mM stock solutions in DMSO and added to culture medium to a final concentration of 10 μM. Media and compounds were refreshed on the fourth day after the initial addition of the compounds. After 8 days of compound incubation, the cells were chemically fixed and stained with anti-acetylated tubulin antibody (Sigma, Cat No. T6793-.5ML), which stains the ciliary axoneme. A fluorescent secondary antibody (Life Technologies, AlexaFluor 488, Cat No. A21145) was used against the primary antibody. The nuclei were counterstained with Hoechst-33258 (Cat No: 382061, Calbiochem). 20 randomized fields per well were imaged at a single plane of focus at 20X magnification in DPBS (Sigma) to reduce auto-fluorescence from the medium and to minimize signal-to-noise ratio. Images acquired by the IN Cell Analyzer 2000 (GE Healthcare) were analyzed using IN Cell Developer software (GE Healthcare).
4
Immunofluorescence Staining Protocol
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Cells were fixed with 3.7% paraformaldehyde, permeabilized for 15 min using 0.1% Triton X and blocked in 0.25% BSA before primary antibody incubations. Primary antibodies used are listed in Additional file 1 : Table S48. Cells were incubated for 2 h at room temperature with the appropriate AlexaFluor-488 or AlexaFluor-546 conjugated antibody (1:500, Invitrogen), DAPI, and CellMask Deep Red (Invitrogen). Images were acquired using the IN Cell 2200 automated microscope (GE), and HCA was performed using the IN Cell Developer software (GE).
5
Quantitative Analysis of YAP Localization
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Two thousand cells were seeded in 96-black well plates (Corning). The following day, cells were fixed with 4% paraformaldehyde for 1 hour and permeabilized with 1% triton for 10 minutes. After blocking with 1% bovine serum albumin (BSA)/PBS for 1 hour, the cells were incubated overnight with FITC-conjugated rabbit YAP antibody (Cell Signaling #14729) in 0.1% BSA/PBS at 4°C. After being washed with PBS, cells were incubated 10 mg/mL Hoechst 33342 and Texas Red phalloidin (Invitrogen) in 0.1% BSA/PBS for 1 hour. Cells were washed 3 times with PBS prior to image acquisition. Image acquisition for each well was performed on an IN Cell Analyzer 2000 (GE Healthcare) by using a 10x objective lens. YAP nuclear/cytoplasmic localization was determined with the IN Cell Developer software (GE Healthcare) by using Hoechst staining to define nuclear regions and phalloidin staining to define cells cytoplasm.
6
Quantifying AAV Transduction Efficiency
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To quantify GFP-positive cells and intensity of GFP fluorescence, images were captured on InCell imager (GE Healthcare, Chalfont St Giles, UK) using 20× magnification. For determining transduction efficiencies using automated InCell Developer Software (GE Healthcare) with a gray level range of 0-65535, up to 3,000 cells were counted. Background GFP fluorescence was determined on cell-free area of each treatment or mock-treated cells, and the GFP-positive cell numbers were normalized to above the background values. In order to determine and compare GFP expression intensities for each treatment, the gray values in the GFP-positive cells above the background were averaged. The collected data represent mean values based on triplicate treatments. Statistical analysis of in vitro AAV transduction data was performed by one-way analysis of variance in GraphPad Prism (version 6.05) with each promoter representing a separate group. Multiple comparisons between the means were done using Tukey’s multiple comparison test with 95% confidence interval.
7
Mitochondrial Morphology Analysis Protocol
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Mitochondrial membrane potential and mitochondrial morphological parameters were measured using tetramethlyrhodamine staining of live fibroblasts. Briefly, 24 h after drug treatment, cells were incubated with 80 nM tetramethlyrhodamine and 10 μM Hoescht in phenol red-free media for 1 h. Cells were washed and imaged using the InCell Analyzer 2000 high-content imager (GE Healthcare). Raw images were processed and parameters obtained using a custom protocol in InCell Developer software (GE Healthcare) allowing for segmentation of mitochondria, nuclei and cell boundaries.
8
Tau Aggregation Assay in HEK293 Cells
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The HEK293 cell line stably expressing the microtubule-binding repeat region of tau fused to yellow fluorescent protein (clone 1) was kindly provided by Marc Diamond 54 (link). The cell line stock tested negative for mycoplasma. Cells were plated at a density of 2000 cells/well in 384 well clear black plates (Greiner Bio-One) and immediately treated with unfractionated tau fibril preparations (0–1 μg tau/well). For experiments where lipofectamine 2000 (Thermo Fisher Scientific) used to introduce fibrils, a ratio of 1 μg tau protein: 0.3 uL lipofectamine 2000 was used. Following a 48 h treatment, nuclei were stained in live cells with 0.012 μg/well of Hoescht 33342 (Thermo Fisher Scientific) for 1 h. Images for DAPI and FITC channels from 3 regions per well were captured using an InCell 6000 (GE HealthCare). The images were processed using InCell Developer software (GE HealthCare) with an algorithm developed to identify live cells with intracellular aggregates larger than 0.89 μm2. Greater than 600 cells per well, assayed in triplicate, was analyzed in 3 independent experiments.
9
Quantifying Proliferation in Mesenchymal Stem Cells
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MSCs were seeded at 15 000 cells/cm2 in 96-well plates, cultured for 3 days in MSC growth media, fixed and immunostained for Ki-67 (1:100; SP6, Thermo Fisher Scientific) followed by Alexa Fluor 647-conjugated secondary antibody (1:1 000; Thermo Fisher Scientific). Nuclei were stained with 1 μg/mL DAPI, for 5 min. The respective manufacturers and dilutions are detailed in Additional file 1 : Table SIV. Cells incubated with the secondary antibody alone were used as a negative control. Five biological replicates were performed per condition. Images were acquired in a IN Cell Analyzer 2 000 (GE Healthcare) using a Nikon 20x/0.45 NA Plan Fluor objective and then analyzed using the IN Cell Developer software (GE Healthcare). The total number of nuclei per image was counted, and the Ki-67 positive cells were quantified. Results are presented as the percentage of Ki-67-positive cells per total number of cells (DAPI-positive).
10
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential was measured using tetramethlyrhodamine (TMRM) staining of live fibroblasts. Briefly; 24 h after drug treatment cells were incubated with 80 nM TMRM and 10 μM Hoechst in phenol red free media for 1 h. Cells were washed and imaged using the InCell Analyzer 2000 high content imager (GE Healthcare, Chicago, IL, USA). Raw images were processed, and parameters obtained using a custom protocol in InCell Developer software (GE Healthcare) allowing for segmentation of mitochondria, nuclei and cell boundaries.
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