The recombinant extracellular domains of the Notch ligands hDLL4-Fc (Cat. 10171-H02H, Sino Biologicals Inc.), hJAG1-Fc (Cat. 11648-H02H, Sino Biologicals Inc.) or IgG-Fc (Cat. 10702-HNAH, Sino Biologicals Inc.) were coated on 24-well plates (Corning, P/N 353226) in Fibronectin matrix (Cat. F1141, Sigma). Following an overnight incubation at 4 °C, primary ECs at 80% confluency were trypsinized and seeded onto the coated plates and incubated at 37 °C with 5% CO2 for indicated amount of time. Candidate targets identified by RNAseq were validated by RT-qPCR on a different frozen batch of HUVECs or HRECs.
Fibronectin matrix
Manufactured by Merck Group
Fibronectin matrix is a laboratory product that provides a natural extracellular matrix for cell culture applications. It serves as a substrate for cell attachment, proliferation, and differentiation. The core function of the fibronectin matrix is to mimic the native cellular environment, supporting various in vitro cell-based assays and experiments.
Automatically generated - may contain errors
Lab products found in correlation
Hbmec, by Merck Group (1 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (1 mentions)
Endothelial cell medium (ecm), by ScienCell (1 mentions)
Penicillin, by ScienCell (1 mentions)
Streptomycin, by ScienCell (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
24 well plate, by Corning (1 mentions)
4 protocols using fibronectin matrix
1
Notch Ligand-Mediated Endothelial Cell Assay
2
Culturing Primary hBMEC and hAEC for Binding Assays
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Primary hBMEC (ScienCell Research laboratories, CA, US) and primary hAEC (ATCC) were cultured in endothelial cell medium (ScienCell Research Laboratories) supplemented with 5% FBS, endothelial cell growth supplement (ScienCell Research Laboratories) and Penicillin (100 U/ml) and streptomycin (100 μg/ml) (ScienCell Research Laboratories). hBMEC were cultured on a Fibronectin matrix (Sigma-Aldrich). For hBMEC passage 5-7 was used, while for hAEC passage 4-7 was used in the binding assays.
3
Confocal Imaging of CD4-Fc and CD45+ Cells
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Samples from mice for confocal imaging were prepared as described previously [54 (link)]. Briefly, cells were stained for CD4-Fc protein and with an anti-CD45+ antibody for 1 h. Next, the cells were washed and stained with anti-CD4-Fc secondary antibody for 1 h. Following washing, the cells were transferred onto a fibronectin matrix (Sigma-Aldrich) and fixed in 2% paraformaldehyde. Slides were mounted with #1.5 coverslips and Mowiol mounting medium and examined using an FV3000 confocal microscope.
4
Tethered Ligand Assay for Notch Signaling
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Tethered Ligand Assay: The recombinant extracellular domain of the Notch ligand hDLL4FC (Sino Biologicals Inc.) or IgG-FC (Sino Biologicals Inc.) were coated onto a 24-well plate (Corning) on a fibronectin matrix (Sigma). Following an overnight incubation at 4 °C, primary ECs (at 80% confluency) were trypsinized and seeded onto the coated plates and incubated at 37 °C with 5% CO2 for 6 h. Experiment was performed in triplicate.
RNA was isolated using the RNEasy Mini Kit (Qiagen), quantity and integrity measured using a Bio-analyzer (Agilent TapeStation 4200, UIC Genome Research core) prior to RNA sequencing. TLA HdLEC samples were sequenced at a ~30 million paired-end (PE) read depth with 150-base fragments by Novogene (https://en.novogene.com/ ). Raw reads from in vitro screens were mapped to the Human database (ENSEMBL/GRCh38) using STAR (version 2.5.0a) and processed with Samtools (version 1.4.1). The counts obtained by FeatureCounts (version 1.5.2) were analyzed by DESeq2 (version 1.18.1) to identify differentially expressed genes. The RNAseq datasets generated in the current study are available in the NCBI Gene Expression Omnibus repository at https://www.ncbi.nlm.nih.gov/geo (Accession Number GSE183631).
RNA was isolated using the RNEasy Mini Kit (Qiagen), quantity and integrity measured using a Bio-analyzer (Agilent TapeStation 4200, UIC Genome Research core) prior to RNA sequencing. TLA HdLEC samples were sequenced at a ~30 million paired-end (PE) read depth with 150-base fragments by Novogene (
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