cell basal media mv2  (PromoCell)

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury

doi: 10.1101/2023.06.09.544380

Figure Lengend Snippet: Isolation and Identification of renal lymphatic endothelial cells. A) Diagrammatic outline of lymphatic endothelial cell (LEC) isolation method and follow on bioinformatics analysis. B) Flow cytometric verification of renal lymphatic endothelial cell populations captured with magnetic enrichment as verified by gating of CD45-live cells, PDPN+CD31+, and LYVE1+PDPN+ C) Anchored clustering analysis of renal LEC single cell data sets identifies seven cell populations through canonical correlation analysis (CCA) unsupervised clustering. D) Lymphatic identity of cell populations verified through expression of lymphatic genes.

Article Snippet: Human dermal lymphatic endothelial cells (HDLECS) were purchased from Promocell (C-122217) and maintained in complete endothelial basal media (C-22022, Promocell) at 37°C, 5% CO2 conditions as previously described .

Techniques: Isolation, Expressing

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury

doi: 10.1101/2023.06.09.544380

Figure Lengend Snippet: Genetic signatures of renal lymphatic endothelial cells. A) In control conditions lymphatic endothelial cell (LEC) populations 1 and 2 comprised a bulk of the population and isolated as a subset for LEC specific analyses. In control conditions 458 total LECs were utilized in downstream analyses. Scaled expression level heatmap of top 5 genes from the identified cell clusters. B) UMAP of control isolated cell populations and selected LEC subset (composed of LEC 1 and LEC2) utilized for subsequent downstream analysis. C) Expression level scaled heatmap of top 5 marker genes in LEC subcluster phenotypes. LEC subclusters differentiate into four distinct anatomical locations. D) Previously published gene markers used to identify distinct lymphatic vessel subpopulation LECs. E) Diagrammatic depiction of the anatomical location of captured LEC populations.

Article Snippet: Human dermal lymphatic endothelial cells (HDLECS) were purchased from Promocell (C-122217) and maintained in complete endothelial basal media (C-22022, Promocell) at 37°C, 5% CO2 conditions as previously described .

Techniques: Control, Isolation, Expressing, Marker

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury

doi: 10.1101/2023.06.09.544380

Figure Lengend Snippet: Validation of lymphatic genetic response to kidney injury. A) Immunofluorescent staining of Spp1 (red) and Pdpn (green) demonstrating increased Spp1 upon cisplatin injury and co-localization with Pdpn+ lymphatic vessel (arrow). B) Immunofluorescent staining of Fabp4 (red) and Ppdn (green) with co-localization in injury (arrow). Images collected at 40x on 10uM sections. Scale bar= 20uM. C) Heatmap of log2FC values of isolated renal LECs for highly changed genes as determined by scRNA sequencing data in cisplatin and ischemia reperfusion injury (IRI). Genes are normalized to respective condition controls. D) qPCR relative expression values in cisplatin isolated renal lymphatic endothelial cells (LECs) confirms significant gene expression changes with injury for Ptprb, Mgp, Gng11, and Vim as identified in scRNA sequencing results in . Kdr (p = 0.0575) and both Slc34a1 and Fabp4 were not detected as significantly different. D) qPCR relative expression values in renal LECs isolated from ischemia reperfusion injury (IRI) confirms similar significant gene expression changes with injury for Ptprb and similar trends to cisplatin LECs for Slc34a1 and Kdr. In IRI Fabp4 had significantly decreased relative expression when compared to control conditions. *= p <0.05, **= p <0.005, ****= p <0.0001.

Article Snippet: Human dermal lymphatic endothelial cells (HDLECS) were purchased from Promocell (C-122217) and maintained in complete endothelial basal media (C-22022, Promocell) at 37°C, 5% CO2 conditions as previously described .

Techniques: Biomarker Discovery, Staining, Isolation, Sequencing, Expressing, Gene Expression, Control

Journal: bioRxiv

Article Title: Single-cell RNA sequencing identifies response of renal lymphatic endothelial cells to acute kidney injury

doi: 10.1101/2023.06.09.544380

Figure Lengend Snippet: Gene expression of cisplatin-treated HDLECs in vitro . A) Heatmap of log2FC values of Human Dermal Lymphatic Endothelial Cells (HDLECs) highly changed genes as determined by scRNA sequencing data. HDLECs genes compared in cultures treated for 24 hours with 4 µg/mL or 8 µg/mL cisplatin. Genes are normalized to saline treated controls. B) Relative expression values of Ptprb, Vim, Kdr, Fabp4 and Rsad2 in HDLECs confirming scRNA sequencing gene expression. Spp1 relative gene expression unlike scRNA sequencing expression and immunofluorescence validation. *= p <0.05, **= p <0.005, ***= p<0.0005.

Article Snippet: Human dermal lymphatic endothelial cells (HDLECS) were purchased from Promocell (C-122217) and maintained in complete endothelial basal media (C-22022, Promocell) at 37°C, 5% CO2 conditions as previously described .

Techniques: Gene Expression, In Vitro, Sequencing, Saline, Expressing, Immunofluorescence, Biomarker Discovery