Prior to high content screening (HCS) platform analysis, HCV core protein and SAMHD1 protein in cells were labeled with fluorescence according to immunofluorescence staining. The correlation of fluorescence intensity between SAMHD1 and HCV core protein was detected by the CellInsight CX5 HCS platform (Thermo Fisher Scientific) with a ×10 objective. HCS Studio™ cell analysis software was applied to quantify proteins based on their own signal intensity. A limit was typically set on cells without SAMHD1 overexpressing.
Cellinsight cx5 hcs platform
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellInsight CX5 HCS Platform is a high-content screening system designed for quantitative cellular analysis. The platform provides automated image acquisition, analysis, and data management capabilities for a wide range of cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Echo 555, by Beckman Coulter (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
384 well plate, by Greiner (1 mentions)
Pkh26gl 1kt, by Merck Group (1 mentions)
Poly mono adp ribose, by Cell Signaling Technology (1 mentions)
Multidrop combi, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions)
Dako omnis, by Agilent Technologies (1 mentions)
96 well plate, by PerkinElmer (1 mentions)
Epigenetic compound library, by Selleck Chemicals (1 mentions)
6 protocols using cellinsight cx5 hcs platform
1
Quantifying Protein Colocalization in HCS
2
High-Throughput PARP Inhibitor Screening
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Cells were plated at a density of 6,000 cells per well into 384-well plates (Greiner, Austria; 781090) using a Multidrop Combi. PARPi were added with Echo555 (LabCyte) at final concentration range between 1.6 pmol/L and 30 μmol/L and incubated for 4 hours; Methyl methanesulfonate (MMS; Sigma Aldrich, 129925) was added at final 0.01%. Cell media was removed and a pre-extraction step was performed for 10 min at 4°C with cold cytoskeleton (CSK) buffer (10 mmol/L PIPES pH = 6.8, 300 mmol/L sucrose, 200 mmol/L NaCl, 3 mmol/L MgCl2) supplemented with 0.6% Triton X-100. Cells were then fixed with ice-cold methanol for 15 minutes at −20°C; blocking solution (PBS + 0.1% Tween20 + 3% BSA) was added and incubated for 1 hour at room temperature. Immunostaining of PARP1 or PARP2 was performed by adding the primary antibody (PARP1 antibody, ProteinTech, 13371–1-AP; PARP2 antibody, Active Motif, 39743) diluted 1:1,000 in PBS + 1% BSA and incubated overnight at 4°C, followed by incubation with secondary goat anti-rabbit AlexaFluor 488 antibody for 1 hour at room temperature. DNA was counterstained with Hoechst 33342 (Invitrogen, H3570) diluted 1:5,000 in PBS + 1% BSA. The nuclear F.I. was acquired and analyzed using CellInsight CX5 HCS Platform (Thermo Fisher); dose–response curves were generated with GraphPad Prism Software.
3
Uptake of Extracellular Vesicles by Chondrocytes
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EVs (1.0×109 particles) isolated after exercise were labelled with PKH26 (cat no. PKH26GL-1KT, Sigma) according to the manufacturer’s instructions. Briefly, EVs were incubated with PKH26 for 10 minutes at room temperature. EVs were then washed in wash buffer and centrifuged at 16,100 g for 30 minutes at 4°C. The supernatant was removed until the 100 μL mark and the residual volume was mixed gently. Subsequently, the PKH26-labeled EVs were supplemented to human chondrocytes and incubated for 2 hours under live cell imaging (cat no. CX51110, Cellinsight CX5 HCS Platform, ThermoFisher). Chondrocytes were visualized using MitoTracker.
4
Quantifying PAR Induction by PARPi and H2O2
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Cells were seeded into 384-well plates (Greiner, Austria; 781090) using a Multidrop Combi (Thermo Fisher Scientific) and incubated overnight at 37°C and 5% CO2 in a rotating incubator. PARPi compounds were added with Echo555 (LabCyte), with final compound concentration range between 1.6 pmol/L and 30 μmol/L. After 1 hour incubation with PARPi, H2O2 was added at final concentration of 10 mmol/L and incubated for 5 min in culture conditions. Cells were then fixed in ice-cold methanol for 15 minutes at 4°C and processed for immunostaining with primary antibody Poly/Mono-ADP Ribose (Cell Signaling Technology, #83732) followed by secondary goat anti-rabbit AlexaFluor-488 antibody (Invitrogen, #A-11008); nuclei were counterstained with DAPI. The nuclear PAR fluorescence intensity (F.I.) was acquired and analyzed using CellInsight CX5 HCS Platform (Thermo Fisher Scientific); dose–response curves were generated with GraphPad Prism software.
5
Immunofluorescence and Lysosome Imaging
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For IF staining, MEF cells were cultured on pre-coated cover glasses and subjected to fixation. The glass slides were incubated overnight in the dark with fluorescence-tagged antibodies against NRF2 and TAZ and counterstained with DAPI for 5 min. For LysoTracker staining, the slides were stained with LysoTracker Red DND-99 (50 nM; Thermo Fisher Scientific). The slides were then mounted with a fluorescence mounting medium (DAKO Omnis; Agilent Technologies Inc., Santa Clara, CA, USA) and observed under a confocal laser microscope (Nikon A1R). For the HCS assay, cells were plated onto the HCS-based plates and stained with slides were fluorescence-tagged antibodies against LC3B, followed by HCS platform analysis. Automated cellular imaging and fluorescence intensity of cellular puncta were quantitatively analyzed using the CellInsight CX5 HCS Platform (Thermo Fisher Scientific).
6
Screening Epigenetic Compounds in Megakaryocytic Cells
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The human PF4 promoter-GFP reporter vector, PLVX-PF4-promoter-GFP, was constructed as previously reported [28 (link)]. The megakaryocytic cell lines MEG01 and K562 were transfected with PLVX-PF4-promoter-GFP and selected with puromycin. K562 cells with PLVX-PF4-promoter-GFP were resuspended in culture medium (2 × 104 cells/mL) and aliquoted into 96-well plates (PerkinElmer, Waltham, MA, USA). The compounds were added immediately after plating. The cells were cultured at 37 °C in 5% CO2 for 48 h and analyzed using a Cell Insight CX5 HCS platform (Thermo Fisher Scientific, Waltham, MA, USA) [29 (link)]. An epigenetic compound library composed of 120 chemical compounds was purchased from Selleckchem (Houston, TX, USA).
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