human umbilical cord ecs (huvecs)  (Angiocrine)

Journal: bioRxiv

Article Title: Tumoral hypoxic extracellular vesicles create a protective microenvironment for triple-negative breast cancer

doi: 10.1101/2024.11.01.621519

Figure Lengend Snippet: A - Representative images of HUVEC (nuclei in cyan, F-actin in magenta) treated with PBS or EVh on a gelatin coating (gray), with degradation spots in black (scale bar: 50 µm). B – Quantification of degraded gelatin area (µm 2 ) in individual HUVEC cultures. C – Cell circularity index for quantification of HUVEC morphology in individual culture. D - Tracking of HUVEC trajectory over 24h. E – Average track speed of cells in 24 h (µm/s). F - Total distance traveled by cells in 24 h (µm). G – Experimental design of the indirect co-culture assay, created in BioRender.com. H – Representative images of HUVEC (nuclei in cyan, F-actin in yellow; scale bar: 50 µm) in indirect co-culture with MDA-MB-231 (nuclei in cyan, F-actin in magenta; scale bar: 100 µm). I – Cell circularity index for quantification of HUVEC and MDA-MB-231 morphology in direct co-culture. J – Representative images of gelatin matrix (gray) with degradation spots identified by white arrows. K - MMP-2 detected by gelatin zymography and densitometry analysis to identify gelatinase activity in indirect co-culture. L – Quantification of degraded gelatin area (µm 2 ) in indirect co-culture. M - Experimental design of the direct co-culture assay, created in BioRender.com. N – Representative images of HUVEC (cytoplasm in yellow) in direct co-culture with MDA-MB-231 (nuclei in cyan, F-actin in magenta) seeded atop a gelatin coating (gray) (scale bar: 100 µm). O - Cell circularity index for quantification of combined cell morphology in direct co-culture. P - Quantification of degraded gelatin area (µm 2 ) in direct co-culture. Q – Cell viability of HUVEC and MDA-MB-231 direct co-culture. Assays were repeated on three different occasions with technical triplicates. p values indicated above comparative bars with statistical significance.

Article Snippet: Dermal fibroblasts HDFa (ATCC ® PCS-201-012 TM ) and umbilical cord endothelial cells HUVEC (ATCC ® CRL-1730 TM ) were maintained between passages 6 and 15 in DMEM 10% FBS 1% pen/strep at 37 °C, 5% CO 2 .

Techniques: Co-culture Assay, Co-Culture Assay, Zymography, Activity Assay

Journal: bioRxiv

Article Title: Tumoral hypoxic extracellular vesicles create a protective microenvironment for triple-negative breast cancer

doi: 10.1101/2024.11.01.621519

Figure Lengend Snippet: A - Representative images of HDFa (nuclei in cyan, F-actin in magenta) treated with PBS or EVh on a gelatin coating (gray), with degradation spots identified by white arrows (scale bar: 100 µm). B – Cell circularity index for quantification of HDFa morphology in individual culture. C - Quantification of degraded gelatin area (µm 2 ) in individual HUVEC cultures. D – HDFa cell viability in individual culture. E - Tracking of HDFa trajectory over 24h. F – Average track speed of cells in 24 h (µm/s). G - Total distance traveled by cells in 24 h (µm). H – Representative images of HDFa (nuclei in cyan, F-actin in yellow; scale bar: 100 µm) in indirect co-culture with MDA-MB-231 (nuclei in cyan, F-actin in magenta; scale bar: 30 µm). I – Cell circularity index for quantification of HDFa and MDA-MB-231 morphology in indirect co-culture. J - Representative images of gelatin matrix (gray) with degradation spots identified by white arrows. K - Quantification of degraded gelatin area (µm 2 ) in indirect co-culture. L – MMP-2 detected by gelatin zymography and densitometry analysis to identify gelatinase activity in indirect co-culture. M – Representative images of MDA-MB-231 (cytoplasm in yellow) in direct co-culture with HDFa (nuclei in cyan, F-actin in magenta) seeded atop a gelatin coating (gray) (scale bar: 100 µm). N - Cell circularity index for quantification of combined cell morphology in direct co-culture. O – Quantification of degraded gelatin area (µm 2 ) in direct co-culture. Assays were repeated on three different occasions with technical triplicates. p values indicated above comparative bars with statistical significance.

Article Snippet: Dermal fibroblasts HDFa (ATCC ® PCS-201-012 TM ) and umbilical cord endothelial cells HUVEC (ATCC ® CRL-1730 TM ) were maintained between passages 6 and 15 in DMEM 10% FBS 1% pen/strep at 37 °C, 5% CO 2 .

Techniques: Co-Culture Assay, Zymography, Activity Assay

Journal: bioRxiv

Article Title: Tumoral hypoxic extracellular vesicles create a protective microenvironment for triple-negative breast cancer

doi: 10.1101/2024.11.01.621519

Figure Lengend Snippet: A – Experimental design of the multicellular circulating co-culture assay (MC-CC) using the QV500 system (Kirstall). B - Representative images of MC-CC chambers containing MDA-MB-231, HUVEC or HDFa treated with PBS or EVh. (scale bars: 30 µm and 50 µm). C - Quantification of degraded gelatin area (µm 2 ) in the MC-CC MDA-MB-231 chamber. D – Band densitometry of MMP-2 detected by gelatin zymography from the MC-CC conditioned media. E,F – Cytokines IL-1β and IL-6 probed from the MC-CC conditioned media by ELISA. G,H – Relative fluorescence intensity of β-catenin and VEGFR2 in HUVEC, respectively. I,J – Relative fluorescence intensity of collagen II and III in HDFa, respectively. Assays were repeated on four different occasions with a single replicate. p values indicated above comparative bars with statistical significance.

Article Snippet: Dermal fibroblasts HDFa (ATCC ® PCS-201-012 TM ) and umbilical cord endothelial cells HUVEC (ATCC ® CRL-1730 TM ) were maintained between passages 6 and 15 in DMEM 10% FBS 1% pen/strep at 37 °C, 5% CO 2 .

Techniques: Co-culture Assay, Zymography, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: bioRxiv

Article Title: Tumoral hypoxic extracellular vesicles create a protective microenvironment for triple-negative breast cancer

doi: 10.1101/2024.11.01.621519

Figure Lengend Snippet: A – Representative images of THP-1 (magenta, stained with CellTracker CMPTX) attachment to MDA-MB-231 MC-CC chambers (nuclei in cyan, F-actin in gray; scale bar: 30 µm). B - Representative images of THP-1 cells (magenta, stained with CellTrace CSFE) in direct co-culture with MDA-MB-231, HUVEC or HDFa (nuclei in cyan, F-actin in gray; scale bars: 50 µm). C - Cell circularity index for quantification of combined cell morphology in THP-1 direct co-culture with MDA-MB-231, HUVEC and HDFa. D – Quantification of degraded gelatin area (µm 2 ) in THP-1 direct co-culture with MDA-MB-231, HUVEC and HDFa. Assays were repeated on three different occasions with technical triplicates. p values indicated above comparative bars with statistical significance.

Article Snippet: Dermal fibroblasts HDFa (ATCC ® PCS-201-012 TM ) and umbilical cord endothelial cells HUVEC (ATCC ® CRL-1730 TM ) were maintained between passages 6 and 15 in DMEM 10% FBS 1% pen/strep at 37 °C, 5% CO 2 .

Techniques: Staining, Co-Culture Assay

Journal: eLife

Article Title: Human primed ILCPs support endothelial activation through NF-κB signaling

doi: 10.7554/eLife.58838

Figure Lengend Snippet: ( a ) Circulating human ILCs are identified as lineage negative CD127 + cells; within this population, we discriminate ILC1s as c-Kit - CRTH2 - , ILC2s as CRTH2 + c-Kit +/- , and ILCPs as c-Kit + CRTH2 - cells. HUVEC cells were co-cultured for 3 hr at 1:1 ratio in direct contact with either in vitro-expanded ( b ) or directly ex vivo-sorted ( c ) ILC1s, ILC2s, and ILCPs. Untreated ECs were employed as negative control (CTRL). ECs were harvested and analyzed for cell-surface adhesion molecule expression by flow cytometry. Graphs show representative histograms (panels b and c , top) and the summary (panels b and c , bottom) of the induction of the indicated adhesion molecules on the EC surface (n = 6). Ordinary one-way ANOVA–Tukey’s multiple comparison test (panel b ); Ordinary one-way ANOVA–Friedman test (panel c ). Figure 1—source data 1. Raw data of panels b and c.

Article Snippet: Primary human umbilical cord vein ECs (HUVECs – Lonza) and primary HDBECs (Promocell) were cultured in supplemented EC growth medium (EGM Ready To Use, Lonza) and used between passages 4 and 6.

Techniques: Cell Culture, In Vitro, Ex Vivo, Negative Control, Expressing, Flow Cytometry

Journal: eLife

Article Title: Human primed ILCPs support endothelial activation through NF-κB signaling

doi: 10.7554/eLife.58838

Figure Lengend Snippet: ( a ) The supernatant of the 3 hr co-culture experiments between ECs and ILCPs was analyzed for its cytokine contents (n = 4). The composition of the supernatant of ECs in EC growth medium was used as negative control (CTRL). ( b ) The expression of IL-6, IL-8, GM-CSF, TNF, and IFN-γ was analyzed by qPCR in ECs and ILCPs after being cultured for 3 hr at 1:1 ratio and FACS-sorted according to CD31 expression. Untreated ECs and ILCPs were employed as controls (CTRL). ( c ) HUVEC cells were co-cultured for 3 hr at 1:1 ratio in direct contact with in vitro-expanded ILCPs either in the absence (red dots) or presence (red circles) of a transwell (TW) insert (0.4 μm pore polycarbonate filter) or ( d ) in the presence of pre-conditioned media coming from previous EC–ILCP 3 hr co-cultures. ECs were harvested and analyzed for cell-surface adhesion molecule expression by flow cytometry (n = 6). The dotted lines indicate the level of average expression of adhesion molecules by unstimulated ECs. Statistical tests used: Unpaired t-test (panels a and d ); paired t-test (panel c ). Figure 2—source data 1. Raw data of panels a–d.

Article Snippet: Primary human umbilical cord vein ECs (HUVECs – Lonza) and primary HDBECs (Promocell) were cultured in supplemented EC growth medium (EGM Ready To Use, Lonza) and used between passages 4 and 6.

Techniques: Co-Culture Assay, Negative Control, Expressing, Cell Culture, In Vitro, Flow Cytometry