For throughput compound screening, human iPSCs were dissociated to single cells with TrypLE Express (GIBCO, Thermo Fisher Scientific, Waltham, MA, USA) and were disseminated onto iMatrix‐coated 96‐well plates with StemFit containing 10 μm Y‐27632 (Nacalai Tesque, Kyoto Japan). After 24 h, the culture medium was replaced with fresh StemFit containing compounds for 3 h, and then, iPSCs were infected with SeV carrying the EGFP gene. Multiplicity of infection (MOI) was estimated to 1. After 48 h of incubation, cells were washed twice with PBS and then fixed in 4% paraformaldehyde (PFA) for 10 min at room temperature. 4’6‐Diamidino‐2‐phenylindole (DAPI) (Life Technologies, Waltham, MA. USA) was used to label the nuclei. Cell images were acquired with IN CELL Analyzer 6000 (GE Healthcare, Chicago, IL, USA) in the throughput screening and IN CELL Analyzer 2000 (GE Healthcare) in the dose dependency assay, and the number of EGFP‐positive cells was quantified using IN CELL Developer toolbox software 1.92 (GE Healthcare).
In cell developer toolbox software 1
Manufactured by GE Healthcare
Sourced in United States
The IN CELL Developer toolbox software 1.92 is a software application designed to analyze and process images obtained from cellular imaging experiments. It provides tools for image acquisition, segmentation, and analysis to support researchers in their study of cellular processes and behaviors.
Automatically generated - may contain errors
Lab products found in correlation
In cell analyzer 6000, by GE Healthcare (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
In cell analyzer 2000, by GE Healthcare (1 mentions)
Tryple express, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Nacalai Tesque (1 mentions)
Bortezomib, by Selleck Chemicals (1 mentions)
Mg132, by Selleck Chemicals (1 mentions)
Ssea 4, by Merck Group (1 mentions)
Nanog, by ReproCELL (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Smi 32, by Fortrea (1 mentions)
Islet1, by Developmental Studies Hybridoma Bank (1 mentions)
4 protocols using in cell developer toolbox software 1
1
High-throughput iPSC Screening Assay
2
Proteasome Inhibition Affects iPSC-Derived Neurons
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iPS-MNs of each clone on Day 35 were seeded at 3.0 x 104cells per well on Matrigel-coated 96-well plates for 48 h after treatment with DMSO or 10 μM MG132, or 10 nM bortezomib (Selleck Chemicals, Houston, TX). The nucleus was stained with DAPI. Each clone was analyzed using IN Cell Analyzer 6000 (GE Healthcare) and IN CELL Developer toolbox software 1.92 (GE Healthcare) in randomized 9-field images in each well. Cell counts treated with MG132 or bortezomib were normalized to cell counts treated with DMSO in each well.
3
Immunostaining of iPSCs and iPSNs
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For immunostaining of iPSCs and iPSNs, cells were fixed in 4% paraformaldehyde (pH 7.4) for 30 min at room temperature and rinsed with PBS. The cells were permeabilized in PBS containing 0.2% Triton X-100 for 10 min at room temperature, followed by rinsing with PBS. After blocking with 5% bovine serum albumin for 60 min at room temperature, cells were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: Nanog (1:500; REPROCELL), SSEA-4 (1:1,000; EMD Millipore), βІІІ-tubulin (1:2,000; Cell Signaling Technology), Islet1 (1:200; Developmental Studies Hybridoma Bank), and SMI32 (1:2,500; Covance). After three rinses with PBS, cells were incubated with appropriate Alexa-Fluor-conjugated secondary antibodies for 1 hr at room temperature, and DAPI (Life Technologies) was used to label nuclei. Cell images were acquired with the IN CELL Analyzer 6000 (GE Healthcare). The number of cells was automatically quantified with IN CELL Analyzer 6000 and IN CELL Developer toolbox software 1.92 (GE Healthcare).
4
Quantifying TFG Puncta in MNs
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HB9::GFP-positive MNs or SMI-32-positive neurons were stained with anti-TFG antibody. The area of TFG puncta in cytoplasm was quantified by IN Cell Analyzer 6000 (GE Healthcare) and IN CELL Developer toolbox software 1.92 (GE Healthcare) in randomized 9- or 16-field images in each well.
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