Journal: iScience

Article Title: APOL1 promotes endothelial cell activation beyond the glomerulus

doi: 10.1016/j.isci.2023.106830

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Article Snippet: STEMdiff Endothelial Differentiation Kit , Stem cell Technologies , 08005.

Techniques: Recombinant, Protease Inhibitor, Enzyme-linked Immunosorbent Assay, Caspase-Glo Assay, Viability Assay, Gene Expression, Bicinchoninic Acid Protein Assay, Single-cell Transcriptomics, Clinical Proteomics, Software, Construct, Electroporation

Journal: Cell Reports

Article Title: Long non-coding RNA PCAT19 safeguards DNA in quiescent endothelial cells by preventing uncontrolled phosphorylation of RPA2

doi: 10.1016/j.celrep.2022.111670

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Article Snippet: iPSC-aggregates were differentiated to cardiac organoids (hCOs) using the STEMdiff Cardiomyocyte Differentiation Kit (#05010, STEMCELL Technologies) following the instructions from the supplier. hCOs were then maintained in mixed medium of STEMdiff Cardiomyocyte Maintenance Basal Medium (#05020, STEMCELL Technologies) and Endothelial Cell Growth Medium 2 (#C-22111, PromoCell) at a ratio of 4:1, with medium changes every second day for a further 28 d. Medium was then changed to medium supplemented with 140 nM CTL LNA or PCAT19 LNA for 48 h. hCOs were then fixed with 4% PFA overnight at 4°C.

Techniques: Recombinant, Produced, Virus, Single Cell Gel Electrophoresis, Sample Prep, Knockdown, Control, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

doi: 10.3390/ijms25073635

Figure Lengend Snippet: rDNA-specific integrated iEPCs and the expression of F8 . ( A ) Schematic diagram of the iEPCs differentiation process, which involves a 5 d small molecule induction followed by maintenance in endothelial growth medium. ( B – D ) Cell morphological changes during the differentiation. ( E ) The differentiation efficiency of CD31 and CD34 double positive iEPCs is analyzed using flow cytometry. ( F ) Immunofluorescence of endothelial progenitor cell markers CD31 (red) and CD144 (green), and DAPI (blue) staining the nuclei. ( G ) Immunofluorescence of endothelial markers CD31 (red) and vWF (green) and DAPI (blue) staining the nuclei. ( H ) Relative transcription levels of F8 (exons 23–26) in B6-iEPCs to HA-iEPCs. GAPDH is used as the internal control. ( I , J ) Analysis of the relative transcription levels of F8 (exons 23–26) in the HA-iEPCs and B6-iEPCs. GAPDH is used as the internal control. ( K ) ELISA results for the FVIII antigen levels in the cell lysate of iEPCs. ( L ) ELISA results for the FVIII antigen levels in the cell culture supernatant of iEPCs. *** p < 0.001, * p < 0.05, “ns” means no significant difference. The bars represent the mean values ± SEM.

Article Snippet: After 1 d, the culture medium was switched with StemDiff APEL2 medium (STEMCELL Technologies) containing 6 μM of CHIR99021 (Selleck Chemicals, Houston, TX, USA) for 2 d. Subsequently, the cells were cultured in StemDiff APEL2 medium supplemented with 25 ng/mL bone morphogenetic protein 4 (BMP4; PeproTech, Cranbury, NJ, USA), 50 ng/mL vascular endothelial growth factor (VEGF; PeproTech), and 10 ng/mL bFGF2 for 2 d. After the 5-d induction, iEPCs were generated and sorted using a CD31 MicroBead Kit (Miltenyi Biotec, Cologne, Germany) for further generation of iECs in ECGM-MV2.

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

doi: 10.3390/ijms25073635

Figure Lengend Snippet: rDNA-specific integrated iEPCs and the expression of F8 . ( A ) Schematic diagram of the iEPCs differentiation process, which involves a 5 d small molecule induction followed by maintenance in endothelial growth medium. ( B – D ) Cell morphological changes during the differentiation. ( E ) The differentiation efficiency of CD31 and CD34 double positive iEPCs is analyzed using flow cytometry. ( F ) Immunofluorescence of endothelial progenitor cell markers CD31 (red) and CD144 (green), and DAPI (blue) staining the nuclei. ( G ) Immunofluorescence of endothelial markers CD31 (red) and vWF (green) and DAPI (blue) staining the nuclei. ( H ) Relative transcription levels of F8 (exons 23–26) in B6-iEPCs to HA-iEPCs. GAPDH is used as the internal control. ( I , J ) Analysis of the relative transcription levels of F8 (exons 23–26) in the HA-iEPCs and B6-iEPCs. GAPDH is used as the internal control. ( K ) ELISA results for the FVIII antigen levels in the cell lysate of iEPCs. ( L ) ELISA results for the FVIII antigen levels in the cell culture supernatant of iEPCs. *** p < 0.001, * p < 0.05, “ns” means no significant difference. The bars represent the mean values ± SEM.

Article Snippet: After 1 d, the culture medium was switched with StemDiff APEL2 medium (STEMCELL Technologies) containing 6 μM of CHIR99021 (Selleck Chemicals, Houston, TX, USA) for 2 d. Subsequently, the cells were cultured in StemDiff APEL2 medium supplemented with 25 ng/mL bone morphogenetic protein 4 (BMP4; PeproTech, Cranbury, NJ, USA), 50 ng/mL vascular endothelial growth factor (VEGF; PeproTech), and 10 ng/mL bFGF2 for 2 d. After the 5-d induction, iEPCs were generated and sorted using a CD31 MicroBead Kit (Miltenyi Biotec, Cologne, Germany) for further generation of iECs in ECGM-MV2.

Techniques: Expressing, Flow Cytometry, Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Cell Culture