Journal: Theranostics

Article Title: Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors

doi: 10.7150/thno.45973

Figure Lengend Snippet: Antibody binding and metabolism in living cells in vitro . Representative immunofluorescence images show the binding of selected anti-mouse (A) and anti-human (B) mAb clones to living endothelial cells. (C, D) EC 50 values of anti-mouse (C) and anti-human (D) mAb clones after binding to living cells for 15 min, n=3. (E, F) Quantitative assessment of the disappearance (due to capture, uptake, and elimination) of anti-mouse (E) and anti-human (F) mAb clones in cell cultures, n=3. (G-I) Cytotoxic effects on living cells in vitro , after different incubation times for anti-mouse mAbs at concentrations of 1-1000 ng/mL (G) or 1-2000 ng/mL (H), or (I) anti-human mAbs at 1-1000 ng/mL, n=3. n.s. no significant difference. mAb: monoclonal antibody; HUVEC: human umbilical vein endothelial cells; HDMEC: human dermal microvascular cells; # P <0.01.

Article Snippet: Human endothelial cell lines (passage 7-10), human umbilical vein endothelial cells (HUVEC,) and human dermal microvascular endothelial cells (HDMEC) (PromoCell, Heidelberg, Germany), were cultured in endothelial cell growth medium and endothelial cell growth medium MV2 (PromoCell) respectively.

Techniques: Binding Assay, In Vitro, Immunofluorescence, Clone Assay, Incubation

Journal: Communications Biology

Article Title: CGRP, adrenomedullin and adrenomedullin 2 display endogenous GPCR agonist bias in primary human cardiovascular cells

doi: 10.1038/s42003-021-02293-w

Figure Lengend Snippet: a Signalling bias plots were calculated as ∆∆Log(τ/K A ) for CGRP in the three cell lines, HUVECs, RAMP1 expressing HUVECs and HCMs for each pathway. Values have been normalised to a reference agonist (AM2) and the reference pathway (cAMP) for all three cell lines. b As for ( a ) except the calculated values are for AM. c Log potency ratios (as measured by the accumulation of cAMP) calculated as Log (EC 50 AM2/EC 50 agonist). Data are compiled from , . HUVECs and HUAECs are shown in red and green respectively, HCMs in cyan and RAMP1-HUVECs in blue. d – f Schematic representation of the signalling bias produced by CGPR ( d ), AM ( e ) and AM2 ( f ), and the intracellular ‘signalling codes’ they bring about based on the potencies recorded at individual pathways in HUVECs, RAMP1-HUVECs, and HCMs.

Article Snippet: HUVECs and HUAECs (were both sourced from PromoCell, Germany; C-12250 and C-12252 respectfully) were cultured in Endothelial Cell Growth Media (ECGM) (PromoCell).

Techniques: Expressing, Produced

Journal: Aging (Albany NY)

Article Title: Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells

doi: 10.18632/aging.203931

Figure Lengend Snippet: Isolation and enrichment strategy of primary NK cells from human PBMC. ( A ) Experimental design of the NK cell enrichment strategy. PBMCs were collected from multiple donors (ages 20-42 years old), and NK cells were isolated and enriched. ( B ) Flow cytometry analysis of CD56 and CD16 expression in NK cells before and after enrichment for a representative donor. ( B-i ) Before enrichment, 1.64% of NK cells (0.18% of PBMCs) were CD56 Bright CD16 - and 46.34% of NK cells (2.8% of PBMCs) were CD56 Dim CD16 + NK cells. ( B-ii ) After enrichment, 2.29% of NK cells (0.47% of PBMCs) were CD56 Bright CD16 - and 66.18% of NK cells (12.6% of PBMCs) were CD56 Dim CD16 + NK cells. ( C-i ) Average percentage of CD56 Dim CD16 + NK cell population in PBMCs from five donors. ( C-ii ) Average percentage of CD56 Bright CD16 - NK cell population in PBMCs from five donors. Donor sex and age are indicated in the figure. Statistical analysis performed using paired t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Primary human arterial endothelial cells purchased from Coriell Institute for medical research (AG10770) were maintained in promo cell basal medium MV2 (PromoCell; Cat# C-22221) supplemented with Growth Medium MV 2 Supplement Pack (PromoCell; Cat# C-39221) and assayed within 10 or less passages.

Techniques: Isolation, Flow Cytometry, Expressing

Journal: Aging (Albany NY)

Article Title: Enhanced co-culture and enrichment of human natural killer cells for the selective clearance of senescent cells

doi: 10.18632/aging.203931

Figure Lengend Snippet: Activated primary NK cells selectively eliminate senescent cells. ( A ) Quantitative Realtime PCR was performed to detect the mRNA levels of CCL5 , CXCL9 , and CXCL11 in non-senescent and senescent IMR-90 fibroblasts. The results are presented as mean fold change in NS compared to S samples from two independent experiments performed in triplicate, and error bars represent ±SEM. Statistical analysis performed using unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001. ( B ) NS or S IMR-90 fibroblasts were co-incubated with NK cells for 16 h at T:E ratios of 1:1, 1:2 and 1:3 and cytotoxicity was evaluated by LDH release. The graphs show the mean and S.E. of % LDH release. NS or S ( C-i ) doxorubicin-treated (n=6), ( C-ii ) irradiated (n=3) or ( C-iii ) etoposide-treated (n=3) IMR-90 fibroblasts or ( C-iv ) doxorubicin-treated endothelial cells (n=2) were overlayed with NK cells for 16 hours at T:E ratio of 1:1, and cytotoxicity was evaluated by LDH release. The results are plotted as mean % cytotoxicity for NS and S cells with each experiment performed in at least triplicate. The graphs show mean % LDH release. ( D ) NK cells isolated and enriched from three different individuals were co-cultured with NS or S IMR-90 cells at T:E ratio of 1:1 and cytotoxicity was evaluated by LDH release after 16 hours of co-culture. Experiments were performed in triplicate and the results are plotted as mean % cytotoxicity for NS and S. Donor sex and age are indicated in the figure. Statistical analysis performed using unpaired t test. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Primary human arterial endothelial cells purchased from Coriell Institute for medical research (AG10770) were maintained in promo cell basal medium MV2 (PromoCell; Cat# C-22221) supplemented with Growth Medium MV 2 Supplement Pack (PromoCell; Cat# C-39221) and assayed within 10 or less passages.

Techniques: Incubation, Irradiation, Isolation, Cell Culture, Co-Culture Assay

Journal: Nature Communications

Article Title: Expansion of functional personalized cells with specific transgene combinations

doi: 10.1038/s41467-018-03408-4

Figure Lengend Snippet: Overview of cell types immortalized in this study

Article Snippet: The following media were used for cultivation of fibroblasts: huFib medium (InSCREENeX GmbH), bone marrow stroma cells: huMSC medium (InSCREENeX GmbH), HUVECs: huVEC medium (InSCREENeX GmbH), HMVECs: huMEC medium (InSCREENeX GmbH), LECs: huLEC medium (InSCREENeX GmbH), chondrocytes: huChon medium (InSCREENeX GmbH), and osteoblasts: huOB maintenance medium (InSCREENeX GmbH).

Techniques:

Journal: Nature Communications

Article Title: Expansion of functional personalized cells with specific transgene combinations

doi: 10.1038/s41467-018-03408-4

Figure Lengend Snippet: Hepatocyte cell lines retain key hepatic properties. a Albumin and Glucose-6-phosphatase gene expression analysis of expandable hepatocytes cultured in standard 2D or 3D (9 days of cultivation as spheroids in spinner flasks) conditions and primary hepatocytes (pHep) either cultured in 3D conditions or freshly isolated (expression normalized to Gapdh, n = 3 independent experiments, except for pHep were n = 2 independent experiments, bars represent mean). b Immunofluorescence-based detection of Albumin and E-cadherin in e-mHepA expandable hepatocytes counterstained for nuclei with DAPI. Scale bar, 20 μm. c Glycogen storage in expandable hepatocytes and control fibroblasts as analyzed by Periodic Acid-Schiff staining. Representative bright field micrographs and quantification of multiple field of views are shown. Scale bar, 100 μm (mean, pooled data from n = 3 independent experiments, field of views analyzed in total 35 to 42). d Immunofluorescence micrograph of Albumin and Hnf4α in e-mHepB hepatocytes after 3D culture as spheroids for 9 days. Actin filaments and nuclei were counterstained with phalloidin and DAPI, respectively. Scale bar, 20 µm. e Induction of phase I metabolic enzyme expression in expandable hepatocytes cultured in 2D or 3D conditions and control fibroblasts in response to stimulation with 50 μM Dexamethasone or 2 μM 3-Methylcholanthrene for 72 h (expression normalized to Gapdh, n = 3 independent experiments, except for e-mHepB Cyp1a1 expression were n = 6 independent experiments, bars represent mean). f Luminescence-based detection of Phase I enzymatic activity in expandable hepatocytes and control fibroblasts after cells were treated as indicate above (activity normalized to 10 4 cells, pooled data from n = 2 independent experiments with n = 3 technical replicates each). g Staining of livers of Fah –/– / Rag2 –/– /Il2rg –/– (FRG) mice for FAH and GFP, 90 days after transplantation of GFP-tagged expandable hepatocytes. Scale bar, 100 µm

Article Snippet: The following media were used for cultivation of fibroblasts: huFib medium (InSCREENeX GmbH), bone marrow stroma cells: huMSC medium (InSCREENeX GmbH), HUVECs: huVEC medium (InSCREENeX GmbH), HMVECs: huMEC medium (InSCREENeX GmbH), LECs: huLEC medium (InSCREENeX GmbH), chondrocytes: huChon medium (InSCREENeX GmbH), and osteoblasts: huOB maintenance medium (InSCREENeX GmbH).

Techniques: Gene Expression, Cell Culture, Isolation, Expressing, Immunofluorescence, Control, Staining, Activity Assay, Transplantation Assay

Journal: Nature Communications

Article Title: Expansion of functional personalized cells with specific transgene combinations

doi: 10.1038/s41467-018-03408-4

Figure Lengend Snippet: Comparable phenotype of primary and expanded endothelial cells. a Phase contrast microscopy shows the expandable HUVEC cell line e-hUVEC-2 (MYC, ID1, and ID2). Scale bar 100 µm. b CD31 expression of human endothelial cell populations immortalized with three different gene sets as indicated. c Global gene expression analysis was performed on three different HUVEC lines (e-hUVEC-2, 8 and 9; in duplicate) (3, 4, 5, respectively) and two independent primary HUVEC populations (2) as well as four primary gingiva fibroblast populations (1). Expression data was processed with GeneSpring 11.5.1 software and a Standard Pearson Correlation was determined for each gene versus all other genes. The correlation heatmap depicts the pair-wise correlation coefficient between the given samples and displays the relationship between the different samples. The samples are clustered based on the pair-wise correlation coefficients between all entities. d Phenotypic stability of e-hUVEC-2 cells after 45 and 90 cumulative population doublings was evaluated by CD31 (also known as PECAM1) expression, acetylated LDL uptake, and eNOS activity. Gray fill: antibody isotype control; black outline: stained sample. MFI: median fluorescence intensity. e Immunofluorescence-based detection of CD31 and CD146 (also known as MCAM) in expandable HUVECs counterstained for nuclei with DAPI. Scale bars, 100 µm. f The phenotype and the functionality of cell line e-hUVEC-2 (cumulative population doubling 80) was compared to primary HUVECs based on flow cytometric analysis of CD31, TIE1, TIE2, and CD309 (also known as VEGFR2) expression. Gray fill: antibody isotype control; black outline: stained sample. MFI: median fluorescence intensity. g The angiogenic potential of primary HUVECs and e-hUVEC-2 was determined in vitro by a matrigel tube formation assay. Scale bars, 200 µm. h Spheroids in matrigel of primary HUVEC and e-hUVEC-2 were subcutaneously injected into Rag2 -/- Il2r g −/− mice. After two weeks the implants were dissected and stained for human CD31 (brown color). e-hUVEC-2 organized into human CD31 positive microvessels similar to primary HUVEC. Scale bars, 100 µm

Article Snippet: The following media were used for cultivation of fibroblasts: huFib medium (InSCREENeX GmbH), bone marrow stroma cells: huMSC medium (InSCREENeX GmbH), HUVECs: huVEC medium (InSCREENeX GmbH), HMVECs: huMEC medium (InSCREENeX GmbH), LECs: huLEC medium (InSCREENeX GmbH), chondrocytes: huChon medium (InSCREENeX GmbH), and osteoblasts: huOB maintenance medium (InSCREENeX GmbH).

Techniques: Microscopy, Expressing, Gene Expression, Software, Activity Assay, Control, Staining, Fluorescence, Immunofluorescence, In Vitro, Tube Formation Assay, Injection