Cellinsight cx5 high content screening platform

Manufactured by Thermo Fisher Scientific

Sourced in United States

The CellInsight CX5 High Content Screening Platform is an automated, high-throughput imaging system designed for cell-based assays. It provides rapid, quantitative analysis of cellular phenotypes and properties across multiple experimental conditions. The system combines high-resolution imaging, advanced image analysis, and data management capabilities to enable efficient and reproducible high-content screening workflows.

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34 protocols using cellinsight cx5 high content screening platform

Quantification of Adipocyte Subpopulations

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Plates were imaged using the CellInsight CX5 High Content Screening (HCS) Platform (Thermo Fisher Scientific, Waltham, MA, USA). Parameters were set to first identify individual cells by detection of Hoechst if they met a certain intensity and area. The software was then programmed to capture the total and average intensity of UCP1 and LipidTOX signals based on nuclei identification. These values were then used to set thresholds of events measuring the total percentage of adipocytes, percentage of white adipocytes, and percentage of brown adipocytes. Cells that contained both UCP1 and LipidTox staining were defined as brown adipocytes. Cells that contained only LipidTox were defined as white adipocytes. The sum of brown and white adipocytes was defined as total adipocytes. The total cell number was detected using Hoechst.

Guijas C., To A., Montenegro-Burke J.R., Domingo-Almenara X., Alipio-Gloria Z., Kok B.P., Saez E., Alvarez N.H., Johnson K.A, & Siuzdak G. (2022). Drug-Initiated Activity Metabolomics Identifies Myristoylglycine as a Potent Endogenous Metabolite for Human Brown Fat Differentiation. Metabolites, 12(8), 749.

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High-Content Screening of Cellular Samples

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Slides were fixed with 4% PFA for 10 min at room temperature (RT) and washed with PBS. Slides were imaged using the CellInsight CX5 High Content Screening (HCS) Platform (Thermo Fisher Scientific). The system was programmed to visit each spot on the array, perform autofocus, and acquire DAPI and FITC (GFP). Cell counts and stain intensities were measured using Thermo Fisher Scientific HCS Studio 2.0 Software using the built-in object identification and cell intensity algorithms.

Kumar N., Richter J., Cutts J., Bush K.T., Trujillo C., Nigam S.K., Gaasterland T., Brafman D, & Willert K. (2015). Generation of an expandable intermediate mesoderm restricted progenitor cell line from human pluripotent stem cells. eLife, 4, e08413.

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Time-Dependent Cell Uptake of siRNAs

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Time-dependent
cell uptake for linear, V-, and Y-shaped siRNAs containing one, two,
and three FITC probes was determined using CellInsight CX5 High Content
Screening (HCS) Platform (Thermo Fisher Scientific). Cells were visualized
2, 4, 8, 24, 48, and 72 h post-transfection. Images were analyzed
using Thermo Scientific HCS Studio Cell Analysis software.

Kozuch S.D., Cultrara C.N., Beck A.E., Heller C.J., Shah S., Patel M.R., Zilberberg J, & Sabatino D. (2018). Enhanced Cancer Theranostics with Self-Assembled, Multilabeled siRNAs. ACS Omega, 3(10), 12975-12984.

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VSV-SUDV Neutralization Assay

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Neutralization assays were performed using vesicular stomatitis virus bearing the GP from SUDV instead of its native G glycoprotein (VSV-GP_SUDV). The viral genome encodes an enhanced green fluorescent protein (eGFP), and infection scored by counting fluorescent cells after infection. The protocol for VSV-GP_SUDV production has been described elsewhere [18 (link)]. Experiments were performed in 96-well tissue grade cell culture plates (Corning). At the time point of infection, Vero cells were at 90% confluency. VSV-GP_SUDV was pre-incubated with the indicated concentrations of antibody or antibody fragments for 15 min. The antibody-virus mixtures were then added in triplicates to the Vero cells. Infection was carried out overnight. At the next day, cells were fixed, stained with DAPI and forwarded to automatic infection counting using the CellInsight CX5 High Content Screening (HCS) platform (ThermoFisher). The percentage of infected cells (green) were calculated in percentage to the DAPI positive cells.

Hofmann D., Zak S.E., Nyakatura E.K., Mittler E., Bakken R.R., Chandran K., Dye J.M, & Lai J.R. (2017). Mechanistic and Fc requirements for inhibition of Sudan virus entry and in vivo protection by a synthetic antibody. Immunology letters, 190, 289-295.

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Quantification of Lipid Accumulation in hASCs

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Lipid accumulation was quantified at differentiation day 9. hASCs differentiated in vitro were washed with PBS and fixed with 4% paraformaldehyde solution for 10 min at room temperature. Fixed cells were washed with PBS and stained with Bodipy 493/503 (0.2 µg/mL; Molecular Probes, Thermo Fisher Scientific) and Hoechst 33342 (2 µg/mL; Molecular Probes) for 20 min at room temperature. After washing with PBS, accumulation of intracellular lipids (Bodipy) and cell number (Hoechst) were quantified with CellInsight™ CX5 High Content Screening (HCS) Platform (Thermo Fischer Scientific). Total Bodipy fluorescence (lipid droplets) was normalized to the amount of nuclei in each well.

Kulyté A., Kwok K.H., de Hoon M., Carninci P., Hayashizaki Y., Arner P, & Arner E. (2019). MicroRNA-27a/b-3p and PPARG regulate SCAMP3 through a feed-forward loop during adipogenesis. Scientific Reports, 9, 13891.

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Quantifying Lipid Accumulation in hMSCs

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Lipid accumulation was quantified at differentiation days 10 and 13. hMSCs differentiated in vitro were washed with PBS and fixed with 4% paraformaldehyde solution for 10 min at room temperature. The fixed cells were subsequently washed with PBS and stained with BODIPY 493/503 (0.2 μg/mL; Molecular Probes, Thermo Fisher Scientific) and Hoechst 33,342 (2 μg/mL; Molecular Probes) for 20 min at room temperature. Accumulation of intracellular lipids (BODIPY) and cell numbers (Hoechst) were quantified with a CellInsight CX5 High Content Screening (HCS) Platform (Thermo Fischer Scientific) using integrated protocols. Total BODIPY fluorescence (lipid droplets) was normalized to the number of nuclei in each well.

Kulyté A., Lundbäck V., Lindgren C.M., Luan J., Lotta L.A., Langenberg C., Arner P., Strawbridge R.J, & Dahlman I. (2020). Genome-wide association study of adipocyte lipolysis in the GENetics of adipocyte lipolysis (GENiAL) cohort. Molecular Metabolism, 34, 85-96.

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Quantifying CRISPR Delivery Efficiency

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Post-transfection efficiency of eGFP-tagged CRISPR/Cas RNP delivery will be assessed using CellInsight™ CX5 High Content Screening (HCS) Platform (Thermo Fisher Scientific) where NucBlue™ Live ReadyProbes™ Reagent (Thermo Fisher Scientific) will act as nuclear counterstain and Wheat Germ Agglutinin, Alexa Fluor™ 647 Conjugate (Thermo Fisher Scientific)–as plasma membrane counterstain. At each time-point (4-, 8-, 24-, 48-, 72- and 96-hours post-transduction) NucBlue® Live reagent (2 drops/mL) and wheat germ agglutinin conjugate (1–10 μg/mL) will be added to the wells of culture plates and incubated for 10–30 minutes. Cells will be then imaged on CellInsight™ CX5 with the following protocol settings:
Nine fields will be analyzed for each well. After CellInsight™ CX5 run will be complete obtained data will be exported to “CSV” file. Total intensity (TotalIntenCh3) gained from channel 3 (green) will reflect CRISPR/Cas RNP delivery efficacy. Obtained data will be analyzed using GraphPad Prism™ 9 Software.

Tyumentseva M.A., Tyumentsev A.I, & Akimkin V.G. (2021). Protocol for assessment of the efficiency of CRISPR/Cas RNP delivery to different types of target cells. PLoS ONE, 16(11), e0259812.

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Quantifying FUS-induced Stress Granule Formation

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Briefly, cells were fixed 24 or 48 h (when FUS is co-transfected with siXPO1) after transfection in 4% formaldehyde in phosphate buffered saline (PBS) and rinsed three times. XPO1 staining was performed as described previously (35 (link)). We blocked the cells in wash buffer (PBS, 0.1% Tween-20 and 2 mg/ml Heparin) supplemented with 3% donkey serum and 5% glycine for 1 h. Subsequently, cells were incubated with the rabbit anti-mouse CRM1 antibody (Novus Biologicals, Littleton, CO, USA; NBP2–16014), diluted 1:100, for 16 h at 4°C. After washing the cells four times with wash buffer, Alexa Fluor-647 conjugated secondary antibodies (Invitrogen) were applied to the cells for 3 h at RT. Secondary antibodies were diluted 1:1000 in wash buffer with 3% donkey serum. By confocal microscopy, we analyzed the localization of XPO1. In order to determine the recruitment of FUS into cytoplasmic granules, cells were analyzed using the CellInsight™ CX5 High Content Screening (HCS) Platform (ThermoFisher Scientific; Cat#CX51110). Nuclear and cytoplasmic intensities were analyzed with the HCS studio cell analysis software (ThermoFisher Scientific). Stress granules were induced by incubating the cells for 1 h with 0.5 mm NaAsO₂ (Sigma).

Steyaert J., Scheveneels W., Vanneste J., Van Damme P., Robberecht W., Callaerts P., Bogaert E, & Van Den Bosch L. (2018). FUS-induced neurotoxicity in Drosophila is prevented by downregulating nucleocytoplasmic transport proteins. Human Molecular Genetics, 27(23), 4103-4116.

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Quantifying Lipid Accumulation and Lipolysis in hMSCs

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Lipid accumulation was quantified at differentiation day 9. hMSCs differentiated in vitro were washed with PBS and fixed with 4% paraformaldehyde solution for 10 min at room temperature. Following fixation, neutral lipids were stained with Bodipy 493/503 (at 0.2 µg/mL; Molecular Probes, Thermo Fisher Scientific, Waltham, MA, US), and nuclei (DNA) were stained with Hoechst 33342 (at 2 µg/mL; Molecular Probes) for 20 min at room temperature. Accumulation of neutral lipids and cell numbers were quantified in CellInsight™ CX5 High Content Screening (HCS) Platform (Thermo Fischer Scientific, Waltham, MA, US) with integrated “Spot detection” protocol. Total Bodipy fluorescence (lipid droplets) was normalized to the number of nuclei, representing the number of cells in each well.
The medium was collected at day 7 and 9 of differentiation of hMCSs for measuring glycerol release as an index of lipolysis, as described [27 (link)]. A standard curve ranging from 0 to 120 µM was used to calculate the concentrations of the samples. Amounts of glycerol was normalized to the number of nuclei and in each well.

Lundbäck V., Kulyté A., Arner P., Strawbridge R.J, & Dahlman I. (2020). Genome-Wide Association Study of Diabetogenic Adipose Morphology in the GENetics of Adipocyte Lipolysis (GENiAL) Cohort. Cells, 9(5), 1085.

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Quantifying Proliferation of hASCs

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hASCs were electroporated at proliferation stage (day −4 before initiation of differentiation). Two days posttransfection (day −2), the cells were incubated with media containing 5 μmol/L 5-ethynyl-2′-deoxyuridine (EdU) for 24 h. EdU-positive cells and total cell number (nuclear staining) were assessed with the Click-iT Plus EdU Alexa Fluor 555 kit (C10352; Invitrogen) according to manufacturer protocols. Rate of proliferation (EdU-positive cells) was normalized to the number of nuclei representing number of cells in each well. Quantification of cells was performed with CellInsight CX5 High Content Screening (HCS) Platform (Thermo Fischer Scientific, Waltham, MA) with integrated “Object detection” protocol.

Kulyté A., Aman A., Strawbridge R.J., Arner P, & Dahlman I.A. (2022). Genome-Wide Association Study Identifies Genetic Loci Associated With Fat Cell Number and Overlap With Genetic Risk Loci for Type 2 Diabetes. Diabetes, 71(6), 1350-1362.

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