Structured Review

Becton Dickinson rat monoclonal anti mouse cd29
Reduced β1 integrin activity in amoeboid transition. a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb <t>CD29)</t> expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P
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1) Product Images from "Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis"

Article Title: Calpain-2 regulates hypoxia/HIF-induced amoeboid reprogramming and metastasis

Journal: bioRxiv

doi: 10.1101/2020.01.06.892497

Reduced β1 integrin activity in amoeboid transition. a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb CD29) expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P
Figure Legend Snippet: Reduced β1 integrin activity in amoeboid transition. a, In-silico modelling of cell elongation. Individual cell migration in dependence of friction and contractility using a two-dimensional phase field simulation. b, Active and total β1 integrin protein content (2D monolayer culture). D, densitometric analysis (representative Western blot, n=2-3). c, Schematic of cell isolation for harvesting attached (highly adhesive) and detached (weakly adhesive) cells in 2D culture. d, Active and total β1 integrin surface expression (MFI) in 4T1 subpopulations 48 h after treatment (left panel, representative flow cytometry histogram). Ratio of active/total β1 integrin surface expression normalized to vehicle control ratios (right panel). Columns show the median from independent experiments (data points). ** P=0.007, * P=0.03 (unpaired t-test, two-sided). e, Confocal micrographs of active (mAb 9EG7) and total β1 integrin (mAb CD29) expression of 4T1 tumoroids invading into 3D collagen (left panel) and the ratio of active/total β1 integrin per invading 4T1 single cell (right panel, 106 cells). Data show ratios of single cells; horizontal lines the median. Insets, single cell invasion phenotypes (arrowheads). Scale bars, 100 μm (overview), 10 µm (inset). **** P

Techniques Used: Activity Assay, In Silico, Migration, Western Blot, Cell Isolation, Expressing, Flow Cytometry

2) Product Images from "Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow"

Article Title: Discrete populations of isotype-switched memory B lymphocytes are maintained in murine spleen and bone marrow

Journal: bioRxiv

doi: 10.1101/825224

Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. a) Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and BM cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b) Percentage of cells per cluster in each organ by mouse. c) Distribution of IgG subclass mapped on tSNE. d) Signature genes for each cluster, area under curve (AUC) of responder-operator characteristics (ROC) of > 0.7. e) Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. f) Flow-cytometric evaluation of expression of representative markers to identify clusters observed by transcriptional profiles in IgG+ memory B cells (IgG1+ or IgG2b+CD19+CD38+IgM-IgD-CD138-GL7-CD93-Zombie Aqua-small lymphocytes). Dotplots represent one of three mice.
Figure Legend Snippet: Heterogeneity of switched memory B cells is differentially represented in spleen and bone marrow. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. a) Six transcriptionally defined clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and BM cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, presentation is separated by organ. b) Percentage of cells per cluster in each organ by mouse. c) Distribution of IgG subclass mapped on tSNE. d) Signature genes for each cluster, area under curve (AUC) of responder-operator characteristics (ROC) of > 0.7. e) Distribution of transcription levels for representative genes mapped on tSNE. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes. f) Flow-cytometric evaluation of expression of representative markers to identify clusters observed by transcriptional profiles in IgG+ memory B cells (IgG1+ or IgG2b+CD19+CD38+IgM-IgD-CD138-GL7-CD93-Zombie Aqua-small lymphocytes). Dotplots represent one of three mice.

Techniques Used: Sequencing, Expressing, Mouse Assay

a) Experimental setup for the comparison of repertoire of isotype-switched memory B cells of BM and spleen: after isolation, cells of the same organ were divided into equal proportions and processed as biological replicates. After RNA isolation, samples were split and processed as technical replicates. b) Cosine similarity between samples of IgG1/2+ (upper panel) and IgA+ (lower panel) heavy chain CDR3 repertoires accounting for clonotype frequencies. Graphs represent cosine similarity comparison within technical replicates of spleen and BM (blue), within cellular replicates from spleens (red), and between spleen and bone marrow (BM-Spleen, purple) of three individual mice. p values (Welch’s test for difference of means of cosine similarity within shared IgH repertoire (spleen cellular replicates) and between spleen and bone marrow replicates are indicated.
Figure Legend Snippet: a) Experimental setup for the comparison of repertoire of isotype-switched memory B cells of BM and spleen: after isolation, cells of the same organ were divided into equal proportions and processed as biological replicates. After RNA isolation, samples were split and processed as technical replicates. b) Cosine similarity between samples of IgG1/2+ (upper panel) and IgA+ (lower panel) heavy chain CDR3 repertoires accounting for clonotype frequencies. Graphs represent cosine similarity comparison within technical replicates of spleen and BM (blue), within cellular replicates from spleens (red), and between spleen and bone marrow (BM-Spleen, purple) of three individual mice. p values (Welch’s test for difference of means of cosine similarity within shared IgH repertoire (spleen cellular replicates) and between spleen and bone marrow replicates are indicated.

Techniques Used: Isolation, Mouse Assay

The bone marrow contains a major population of isotype-switched non-proliferating memory B cells. a) Quantification of NP-specific IgG2a/b+ spleen, peripheral lymph nodes, blood and BM memory B cells. C57BL/6 mice were immunized with NP-KLH/LPS SC. Numbers of NP-binding+ IgG2b+ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) were determined by flow-cytometry on d421 or d426 post immunization; data pooled from 2 independent experiments. OVA ctrl indicates staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes. Lines connect samples from one individual, p value of paired t test for spleen and BM samples is indicated. b) Flow-cytometric quantification of Ki-67 expression in IgG2b+ Bsm (IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes) splenic naïve (IgM+IgD+IgG2b-CD19+CD38+CD138-GL7-CD11c-PI-small lymphocytes) and germinal center (GC) (CD19+CD38loGL7+CD11c--PI-lymphocytes) B cells. Indicated are the frequencies of Ki-67+ cells within the subset indicated, data in right graph from 2 independent experiments using pooled cells from 4-20 C57BL/6 mice, p value (paired t test) as indicated. c) Flow-cytometric quantification of CD19+ B cells and IgG2b+ memory B cells in mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3x NP-CGG/IFA. Anaylsis was performed after 7 days of CyP. IgG2b+ B cells were quantified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, CD19+ B cells as CD19+CD138-PI-lymphocytes, p value (Welch’s test). Representative data shown for one out of two independent experiments. d) IgG2b+ B memory cells (Ki-67-IgD-Blimp1-GFP-) are dispersed as single cells throughout the bone marrow. Arrows indicate IgG2b+DAPI+ cells. Scale bar: 20µm. e) Co-localization of IgG2b+GFP-IgD-IgG2b+ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b+DAPI+ cells. Representative micrograph. Scale bars: 10µm. f) Co-localization of IgG2b+ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b+ cells in direct contact (black) or within 10mm (grey) of a cell stained for the molecule indicated. g) Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b+ Bsm. Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, histogram plots are representative of three biological replicates.
Figure Legend Snippet: The bone marrow contains a major population of isotype-switched non-proliferating memory B cells. a) Quantification of NP-specific IgG2a/b+ spleen, peripheral lymph nodes, blood and BM memory B cells. C57BL/6 mice were immunized with NP-KLH/LPS SC. Numbers of NP-binding+ IgG2b+ cells in Spleen, BM, blood, and peripheral lymph nodes (pLN) were determined by flow-cytometry on d421 or d426 post immunization; data pooled from 2 independent experiments. OVA ctrl indicates staining controls from mice immunized with the irrelevant antigen ovalbumin (OVA). Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes. Lines connect samples from one individual, p value of paired t test for spleen and BM samples is indicated. b) Flow-cytometric quantification of Ki-67 expression in IgG2b+ Bsm (IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes) splenic naïve (IgM+IgD+IgG2b-CD19+CD38+CD138-GL7-CD11c-PI-small lymphocytes) and germinal center (GC) (CD19+CD38loGL7+CD11c--PI-lymphocytes) B cells. Indicated are the frequencies of Ki-67+ cells within the subset indicated, data in right graph from 2 independent experiments using pooled cells from 4-20 C57BL/6 mice, p value (paired t test) as indicated. c) Flow-cytometric quantification of CD19+ B cells and IgG2b+ memory B cells in mice treated with Cyclophosphamide (CyP) or untreated controls (PBS) after immunization with 3x NP-CGG/IFA. Anaylsis was performed after 7 days of CyP. IgG2b+ B cells were quantified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, CD19+ B cells as CD19+CD138-PI-lymphocytes, p value (Welch’s test). Representative data shown for one out of two independent experiments. d) IgG2b+ B memory cells (Ki-67-IgD-Blimp1-GFP-) are dispersed as single cells throughout the bone marrow. Arrows indicate IgG2b+DAPI+ cells. Scale bar: 20µm. e) Co-localization of IgG2b+GFP-IgD-IgG2b+ cells (arrows) with mesenchymal stromal cells. Arrows indicate IgG2b+DAPI+ cells. Representative micrograph. Scale bars: 10µm. f) Co-localization of IgG2b+ cells to mesenchymal stromal cells. Graph shows frequency of IgG2b+ cells in direct contact (black) or within 10mm (grey) of a cell stained for the molecule indicated. g) Flow cytometric quantification of surface expression of the VLA-4 and VLA-6 components CD29, CD49d, CD49f in spleen and BM IgG2b+ Bsm. Gated for IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes, histogram plots are representative of three biological replicates.

Techniques Used: Mouse Assay, Binding Assay, Flow Cytometry, Staining, Expressing, Immunofluorescence

a) Six transcriptionally defined Clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and bone marrow (BM) cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, representation combined for spleen and BM (left). Distribution of expression for Cd19 , Ptprc (CD45), Pax5 , Cd38 , Sdc1 (CD138), and Bcl6 genes mapped on tSNE representation. b) Distribution of expression for Itgax (CD11c), Itgam (CD11b), Cd5 genes mapped on tSNE representation. c) Single cell gene set enrichment analysis of expression of the Reactome and Gene ontology enrichment (GO) mapped on tSNE representation. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes.
Figure Legend Snippet: a) Six transcriptionally defined Clusters were identified by shared nearest neighbor (SNN) modularity optimization based clustering algorithm mapped to tSNE representation of spleen and bone marrow (BM) cells. tSNE coordinates and clustering was computed for 4754 from spleen and 2947 from BM cells, representation combined for spleen and BM (left). Distribution of expression for Cd19 , Ptprc (CD45), Pax5 , Cd38 , Sdc1 (CD138), and Bcl6 genes mapped on tSNE representation. b) Distribution of expression for Itgax (CD11c), Itgam (CD11b), Cd5 genes mapped on tSNE representation. c) Single cell gene set enrichment analysis of expression of the Reactome and Gene ontology enrichment (GO) mapped on tSNE representation. Cells for single cell sequencing were FACSorted as IgG-expressing CD19+CD38+CD138-GL7-small lymphocytes.

Techniques Used: Expressing, Sequencing

a) Percentage of shared clones between clusters of spleen and BM. Clones were defined according to IgG heavy and light chain sequences. Overlap significantly higher than expected for random overlap (p
Figure Legend Snippet: a) Percentage of shared clones between clusters of spleen and BM. Clones were defined according to IgG heavy and light chain sequences. Overlap significantly higher than expected for random overlap (p

Techniques Used: Clone Assay

Gating for isotype-switched memory B cells of bone marrow and spleen. BM (a) or spleen (b) CD19+ cells were isolated by MACS technology and switched-memory B cells were identified by expression of surface IgG2b, IgG1, or IgA and CD19, CD38 and lack of IgD, IgM, CD138 and GL7 marker. Staining shown for IgG2b exemplarily. c) Cell numbers of Ig-switched memory B cells per organ in C57BL/6 laboratory mice. Absolute cell numbers per organ calculated from flow cytometric counts (gated for IgG1+, IgG2b+, or IgA+ CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes); n=42, data pooled from 8 experiments with five different immunizations protocols performed in C57BL/6 mice aged 4-20 months and held under SPF conditions, data presented as median cell count per organ by immunization. BM: bone marrow, pLN: peripheral lymph nodes, mLN: mesenteric lymph nodes, PP: Peyer’s patches. d) Cell numbers of Bsm per organ in wild and pet shop mice. Median cell numbers per organ calculated from flow cytometric counts (gated for IgG1+, IgG2b+, or IgA+ CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes); n=13 (wild) and 13 (pet). e) Spleen weight from spleens of C57BL/6 mice held under SPF conditions, pet shop and wild mice, n=10 (C57BL/6), 13 (pet), and 11 (wild). f) Identification of bone marrow IgG2b+ Bsm. Naive B cells and plasma cells were excluded by IgD staining and Blimp1-GFP signal, respectively. Cell nucleus was identified with DAPI (blue). Scale bar: 10µm. g) Simulation of co-localization between Bsm and VCAM-1+ stromal cells. Non-random co-localization of bone marrow Bsm was determined using images acquired from 7 bone marrow slides. Graphs represent direct co-localization of more than 12000 simulated cells (random) versus co-localization observed per slide for 28 slides from 4 mice with two or more analyzed Bsm per slide (mean=5.66 cells per slide), p value (Welch’s test) indicated on graph. h) Control stainings for spleen and bone marrow (BM) memory B cells in flow cytometry, identified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes.
Figure Legend Snippet: Gating for isotype-switched memory B cells of bone marrow and spleen. BM (a) or spleen (b) CD19+ cells were isolated by MACS technology and switched-memory B cells were identified by expression of surface IgG2b, IgG1, or IgA and CD19, CD38 and lack of IgD, IgM, CD138 and GL7 marker. Staining shown for IgG2b exemplarily. c) Cell numbers of Ig-switched memory B cells per organ in C57BL/6 laboratory mice. Absolute cell numbers per organ calculated from flow cytometric counts (gated for IgG1+, IgG2b+, or IgA+ CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes); n=42, data pooled from 8 experiments with five different immunizations protocols performed in C57BL/6 mice aged 4-20 months and held under SPF conditions, data presented as median cell count per organ by immunization. BM: bone marrow, pLN: peripheral lymph nodes, mLN: mesenteric lymph nodes, PP: Peyer’s patches. d) Cell numbers of Bsm per organ in wild and pet shop mice. Median cell numbers per organ calculated from flow cytometric counts (gated for IgG1+, IgG2b+, or IgA+ CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes); n=13 (wild) and 13 (pet). e) Spleen weight from spleens of C57BL/6 mice held under SPF conditions, pet shop and wild mice, n=10 (C57BL/6), 13 (pet), and 11 (wild). f) Identification of bone marrow IgG2b+ Bsm. Naive B cells and plasma cells were excluded by IgD staining and Blimp1-GFP signal, respectively. Cell nucleus was identified with DAPI (blue). Scale bar: 10µm. g) Simulation of co-localization between Bsm and VCAM-1+ stromal cells. Non-random co-localization of bone marrow Bsm was determined using images acquired from 7 bone marrow slides. Graphs represent direct co-localization of more than 12000 simulated cells (random) versus co-localization observed per slide for 28 slides from 4 mice with two or more analyzed Bsm per slide (mean=5.66 cells per slide), p value (Welch’s test) indicated on graph. h) Control stainings for spleen and bone marrow (BM) memory B cells in flow cytometry, identified as IgG2b+CD19+CD38+CD138-GL7-CD11c-IgM-IgD-PI-small lymphocytes.

Techniques Used: Isolation, Magnetic Cell Separation, Expressing, Marker, Staining, Mouse Assay, Cell Counting, Positron Emission Tomography, Flow Cytometry

Spleen and bone marrow isotype-switched memory B cells are distinct in Ig heavy chain repertoire. a) and c) Observed overlap between the IgG1/2+ or IgA+ heavy chain CDR3 repertoire of switched memory B cells from spleen and bone marrow or b and d) random distribution (upper panels). Venn diagrams represent clonotype presence in a given sample: numbers indicate clonotypes present in one organ exclusively or in both (overlap). Random distributions (b and d) show median values for 1000 random distributions drawn while retaining the number of clones initially observed in the sample. Histograms (b and d, lower panels) represent number of clonotypes in spleen and BM in 1000 randomized distributions of observed clonotypes to spleen and BM, dashed red line indicates experimentally observed number of clonotypes present in both spleen and BM. P value of one-sided t test for difference of randomized overlap against observed. e) VH gene recruitment to spleen and bone marrow IgG1/2+ (upper panel) and IgA+ (lower panel) memory B cells, represented as frequency of a particular VH gene among total CDR3s per organ. Bars show relative abundance of the 10 most abundant VH genes, error bars indicate SEM. Significance of difference in VH gene distribution to Spleen and BM assessed by MANOVA, p values corrected for multiple testing (Benjamini-Hochberg), * indicates significant difference in means for a particular VH gene (Welch’s test). BM: bone marrow, M1-M3: replicate samples of three C57BL/6 mice immunized 3x NP-CGG/IFA. Only clones consistently found in technical replicates were considered.
Figure Legend Snippet: Spleen and bone marrow isotype-switched memory B cells are distinct in Ig heavy chain repertoire. a) and c) Observed overlap between the IgG1/2+ or IgA+ heavy chain CDR3 repertoire of switched memory B cells from spleen and bone marrow or b and d) random distribution (upper panels). Venn diagrams represent clonotype presence in a given sample: numbers indicate clonotypes present in one organ exclusively or in both (overlap). Random distributions (b and d) show median values for 1000 random distributions drawn while retaining the number of clones initially observed in the sample. Histograms (b and d, lower panels) represent number of clonotypes in spleen and BM in 1000 randomized distributions of observed clonotypes to spleen and BM, dashed red line indicates experimentally observed number of clonotypes present in both spleen and BM. P value of one-sided t test for difference of randomized overlap against observed. e) VH gene recruitment to spleen and bone marrow IgG1/2+ (upper panel) and IgA+ (lower panel) memory B cells, represented as frequency of a particular VH gene among total CDR3s per organ. Bars show relative abundance of the 10 most abundant VH genes, error bars indicate SEM. Significance of difference in VH gene distribution to Spleen and BM assessed by MANOVA, p values corrected for multiple testing (Benjamini-Hochberg), * indicates significant difference in means for a particular VH gene (Welch’s test). BM: bone marrow, M1-M3: replicate samples of three C57BL/6 mice immunized 3x NP-CGG/IFA. Only clones consistently found in technical replicates were considered.

Techniques Used: Clone Assay, Mouse Assay, Immunofluorescence

3) Product Images from "Transplantation of cultured dental pulp stem cells into the skeletal muscles ameliorated diabetic polyneuropathy: therapeutic plausibility of freshly isolated and cryopreserved dental pulp stem cells"

Article Title: Transplantation of cultured dental pulp stem cells into the skeletal muscles ameliorated diabetic polyneuropathy: therapeutic plausibility of freshly isolated and cryopreserved dental pulp stem cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-015-0156-4

Morphology and identification of cryo-DPSCs. a DPSCs, which were thawed after cryopreservation, were cultured and expanded. Cryo-DPSCs were of similar shape as the fresh-DPSCs observed with the phase contrast microscope. Bar = 100 μm. b Flow cytometric analysis of cryo-DPSCs. The expression of surface markers was analyzed with CD29, CD34, CD49d, CD45 and CD90. Open histograms, isotype controls; filled histograms, stained with the specific surface marker antibodies
Figure Legend Snippet: Morphology and identification of cryo-DPSCs. a DPSCs, which were thawed after cryopreservation, were cultured and expanded. Cryo-DPSCs were of similar shape as the fresh-DPSCs observed with the phase contrast microscope. Bar = 100 μm. b Flow cytometric analysis of cryo-DPSCs. The expression of surface markers was analyzed with CD29, CD34, CD49d, CD45 and CD90. Open histograms, isotype controls; filled histograms, stained with the specific surface marker antibodies

Techniques Used: Cell Culture, Microscopy, Flow Cytometry, Expressing, Staining, Marker

Morphology and characterization of fresh-DPSCs. a Cultured DPSCs observed with a phase contrast microscope. Bar = 100 μm. b Flow cytometric analysis of fresh-DPSCs. The expression of the surface markers was investigated with CD29, CD34, CD49d, CD45 and CD90. Open histograms, isotype controls; filled histograms, stained with the specific surface marker antibodies
Figure Legend Snippet: Morphology and characterization of fresh-DPSCs. a Cultured DPSCs observed with a phase contrast microscope. Bar = 100 μm. b Flow cytometric analysis of fresh-DPSCs. The expression of the surface markers was investigated with CD29, CD34, CD49d, CD45 and CD90. Open histograms, isotype controls; filled histograms, stained with the specific surface marker antibodies

Techniques Used: Cell Culture, Microscopy, Flow Cytometry, Expressing, Staining, Marker

4) Product Images from "Mesenchymal stem cells attenuate acute ischemia-reperfusion injury in a rat model"

Article Title: Mesenchymal stem cells attenuate acute ischemia-reperfusion injury in a rat model

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2015.2806

Flow cytometric analyses of the expression of various BMSC markers. BMSCs were cultured for 3 passages and then the cells were lifted with trypsin-free cell detachment buffer. Following incubation with the fluorescence dye-conjugated antibodies specific for CD29, CD73, CD90, CD34 and CD45, the cells were analyzed by flow cytometry. Images are representative of 3–5 independent experiments. BMSCs, bone marrow-derived mesenchymal stem cells; PE, phycoerythrin; FITC, fluorescein isothiocyanate.
Figure Legend Snippet: Flow cytometric analyses of the expression of various BMSC markers. BMSCs were cultured for 3 passages and then the cells were lifted with trypsin-free cell detachment buffer. Following incubation with the fluorescence dye-conjugated antibodies specific for CD29, CD73, CD90, CD34 and CD45, the cells were analyzed by flow cytometry. Images are representative of 3–5 independent experiments. BMSCs, bone marrow-derived mesenchymal stem cells; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Techniques Used: Flow Cytometry, Expressing, Cell Culture, Incubation, Fluorescence, Cytometry, Derivative Assay

Morphology of BMSCs, and CD29 and CD90 expression. (A) BMSCs appeared as fibroblast-like spindle shape under phase-contrast microscopy. The image was captured using a ×20 objective lens. (B) BMSCs were positive for CD90 expression. BMSCs were cultured on chamber slides. Following fixation, the cells were stained with phycoerythrin-conjugated CD90 (dilution, 1:100). (C) BMSCs were positive for CD29 expression. Following fixation, the cells were stained with fluorescein isothiocyanate-conjugated CD29 (dilution, 1:100). (D) Merged image of parts (B) and (C). The fluorescent images were captured using a ×20 objective lens. Images are representative of 3–5 independent experiments. BMSCs, bone marrow-derived mesenchymal stem cells.
Figure Legend Snippet: Morphology of BMSCs, and CD29 and CD90 expression. (A) BMSCs appeared as fibroblast-like spindle shape under phase-contrast microscopy. The image was captured using a ×20 objective lens. (B) BMSCs were positive for CD90 expression. BMSCs were cultured on chamber slides. Following fixation, the cells were stained with phycoerythrin-conjugated CD90 (dilution, 1:100). (C) BMSCs were positive for CD29 expression. Following fixation, the cells were stained with fluorescein isothiocyanate-conjugated CD29 (dilution, 1:100). (D) Merged image of parts (B) and (C). The fluorescent images were captured using a ×20 objective lens. Images are representative of 3–5 independent experiments. BMSCs, bone marrow-derived mesenchymal stem cells.

Techniques Used: Expressing, Microscopy, Cell Culture, Staining, Derivative Assay

5) Product Images from "Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells"

Article Title: Modeling Initiation of Ewing Sarcoma in Human Neural Crest Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0019305

Flow cytometry confirms MSC markers in adherent hNCSC. hNCSC were isolated from in vitro differentiating hESC using p75-FACS. Nearly 50% of the cells also expressed the MSC marker CD73 on Day 0. After 2 days in adherent conditions the morphology of isolated cells changed from small cuboidal cells to larger mesenchymal cells (see Fig. 1A ). Consistent with this morphologic change flow cytometric analysis shows that after 5 days in culture most cells continue to express p75 but, in addition, nearly all cells express the MSC-associated markers CD73, CD105, CD90, CD29, and CD44.
Figure Legend Snippet: Flow cytometry confirms MSC markers in adherent hNCSC. hNCSC were isolated from in vitro differentiating hESC using p75-FACS. Nearly 50% of the cells also expressed the MSC marker CD73 on Day 0. After 2 days in adherent conditions the morphology of isolated cells changed from small cuboidal cells to larger mesenchymal cells (see Fig. 1A ). Consistent with this morphologic change flow cytometric analysis shows that after 5 days in culture most cells continue to express p75 but, in addition, nearly all cells express the MSC-associated markers CD73, CD105, CD90, CD29, and CD44.

Techniques Used: Flow Cytometry, Cytometry, Isolation, In Vitro, FACS, Marker

6) Product Images from "Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum"

Article Title: Multipotency and cardiomyogenic potential of human adipose-derived stem cells from epicardium, pericardium, and omentum

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-016-0343-y

Characterization of human ADSC subsets and BJ fibroblasts ( BJF ). Results of flow cytometric analysis. ADSCs and BJ fibroblasts were positive for the hallmark pattern of MSCs (CD29, CD44, CD90, CD105) and were negative for CD31, CD34, and hematopoietic marker CD45. E/O/P-ADSC epicardial/omental/pericardial adipose-derived stem cell
Figure Legend Snippet: Characterization of human ADSC subsets and BJ fibroblasts ( BJF ). Results of flow cytometric analysis. ADSCs and BJ fibroblasts were positive for the hallmark pattern of MSCs (CD29, CD44, CD90, CD105) and were negative for CD31, CD34, and hematopoietic marker CD45. E/O/P-ADSC epicardial/omental/pericardial adipose-derived stem cell

Techniques Used: Flow Cytometry, Marker, Derivative Assay

7) Product Images from "Inhibition of Wnt/β-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis"

Article Title: Inhibition of Wnt/β-catenin signaling suppresses myofibroblast differentiation of lung resident mesenchymal stem cells and pulmonary fibrosis

Journal: Scientific Reports

doi: 10.1038/s41598-018-28968-9

Mouse lung resident mesenchymal stem cells (LR-MSCs) display MSC properties. ( A ) Mouse LR-MSCs morphology after 7 days of culture following isolation by MACS from the mouse lung single-cell suspension was revealed by a standard light microscope. ( B ) The expression of Sca-1, CD73, CD29, CD44, CD31, CD34 and CD45 on mouse LR-MSCs was measured by flow cytometric analysis. The black line indicates negative control and the red line indicates the respective surface marker staining. ( C ) Sorted mouse LR-MSCs were seeded in an appropriate differentiation-induction medium. Osteogenic differentiation of mouse LR-MSCs was demonstrated by (d) alkaline phosphatase staining at day 7 and (e) alizarin red staining at day 21; Adipogenesis was detected by the formation of lipid droplets stained with oil red O at day 21. (a–c) Control cells were cultured in normal growth medium.
Figure Legend Snippet: Mouse lung resident mesenchymal stem cells (LR-MSCs) display MSC properties. ( A ) Mouse LR-MSCs morphology after 7 days of culture following isolation by MACS from the mouse lung single-cell suspension was revealed by a standard light microscope. ( B ) The expression of Sca-1, CD73, CD29, CD44, CD31, CD34 and CD45 on mouse LR-MSCs was measured by flow cytometric analysis. The black line indicates negative control and the red line indicates the respective surface marker staining. ( C ) Sorted mouse LR-MSCs were seeded in an appropriate differentiation-induction medium. Osteogenic differentiation of mouse LR-MSCs was demonstrated by (d) alkaline phosphatase staining at day 7 and (e) alizarin red staining at day 21; Adipogenesis was detected by the formation of lipid droplets stained with oil red O at day 21. (a–c) Control cells were cultured in normal growth medium.

Techniques Used: Isolation, Magnetic Cell Separation, Light Microscopy, Expressing, Flow Cytometry, Negative Control, Marker, Staining, Cell Culture

8) Product Images from "Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation"

Article Title: Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation

Journal: Scientific Reports

doi: 10.1038/s41598-017-05099-1

CD73 marker identifies the CD29 + CD54 + population. ( a ) Relative mRNA expression of CD73 markers in freshly isolated cells. Gene expression was normalized to that of Hprt (n = 3; *P
Figure Legend Snippet: CD73 marker identifies the CD29 + CD54 + population. ( a ) Relative mRNA expression of CD73 markers in freshly isolated cells. Gene expression was normalized to that of Hprt (n = 3; *P

Techniques Used: Marker, Expressing, Isolation

Morphological and self-renewal evaluation by clonal analysis. ( a ) Representative flow cytometric profiles of rBM cells stained for CD29 and CD54. PI − , CD45 − , and CD31 − cells were gated. Baseline CD29 and CD54 expression is shown in pink. Sorted subsets are shown in black dotted squares. The cell number ratio is shown in the profile. ( b ) Morphological appearance of sorted sub-population cells. Scale bar, 100 µm. ( c ) Single-cell sorting assay of rBM cell sub-populations. WBM cells and four cell populations (CD29 + /CD54 + , CD29 + /CD54 − , CD29 − /CD54 + , and CD29 − /CD54 − ) were sorted into 96-well plates, and the number of colonies formed was counted on day 10. Scale bar, 500 µm.
Figure Legend Snippet: Morphological and self-renewal evaluation by clonal analysis. ( a ) Representative flow cytometric profiles of rBM cells stained for CD29 and CD54. PI − , CD45 − , and CD31 − cells were gated. Baseline CD29 and CD54 expression is shown in pink. Sorted subsets are shown in black dotted squares. The cell number ratio is shown in the profile. ( b ) Morphological appearance of sorted sub-population cells. Scale bar, 100 µm. ( c ) Single-cell sorting assay of rBM cell sub-populations. WBM cells and four cell populations (CD29 + /CD54 + , CD29 + /CD54 − , CD29 − /CD54 + , and CD29 − /CD54 − ) were sorted into 96-well plates, and the number of colonies formed was counted on day 10. Scale bar, 500 µm.

Techniques Used: Flow Cytometry, Staining, Expressing, FACS

CD73 is commonly available for the prospective isolation of MSCs. ( a ) Representative results of flow cytometric analysis for cell surface markers using freshly isolated BMMNCs from rats. The CD73 + population is positive for previously described MSC markers (CD29, CD44, CD54, and CD90). ( b ) Flow cytometric analysis in the CD73 + population for cell surface markers from human and mouse BM (CD29, CD44, CD90, CD140a, CD271, and leptin receptor).
Figure Legend Snippet: CD73 is commonly available for the prospective isolation of MSCs. ( a ) Representative results of flow cytometric analysis for cell surface markers using freshly isolated BMMNCs from rats. The CD73 + population is positive for previously described MSC markers (CD29, CD44, CD54, and CD90). ( b ) Flow cytometric analysis in the CD73 + population for cell surface markers from human and mouse BM (CD29, CD44, CD90, CD140a, CD271, and leptin receptor).

Techniques Used: Isolation, Flow Cytometry

9) Product Images from "Mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model"

Article Title: Mesenchymal stem cell-derived extracellular vesicles attenuate influenza virus-induced acute lung injury in a pig model

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-018-0774-8

Mesenchymal stem cell extracellular vesicles (MSC-EVs) express EV markers. EV-coated latex beads were examined for the expression of EV markers by flow cytometry. EVs expressed the specific EV markers CD9, CD63, and CD81 (broken line: isotype staining; solid line: specific staining)
Figure Legend Snippet: Mesenchymal stem cell extracellular vesicles (MSC-EVs) express EV markers. EV-coated latex beads were examined for the expression of EV markers by flow cytometry. EVs expressed the specific EV markers CD9, CD63, and CD81 (broken line: isotype staining; solid line: specific staining)

Techniques Used: Expressing, Flow Cytometry, Cytometry, Staining

10) Product Images from "Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells"

Article Title: Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0147360

Down-regulation of PSR in HUVECs inhibits their up-taking of MSC-MVs. (A) PSR expression on HUVECs, PSR siRNA-transfected HUVECs (siRNA) and control RNA-treated HUVECs (siRNA-CTR) was evaluated by flow cytometry. The hollow diagrams represent the fluorescence intensity of cells reacted with the FITC-conjugated antibody. (B) The uptake of CFSE-labeled MSC-MVs was observed with flow cytometry. X-axis: the forward scatter corner signals. Y-axis: CFSE intensity. (C)The t-test showed that the PSR expression was significantly decreased by si-RNA (vs.CTR * P
Figure Legend Snippet: Down-regulation of PSR in HUVECs inhibits their up-taking of MSC-MVs. (A) PSR expression on HUVECs, PSR siRNA-transfected HUVECs (siRNA) and control RNA-treated HUVECs (siRNA-CTR) was evaluated by flow cytometry. The hollow diagrams represent the fluorescence intensity of cells reacted with the FITC-conjugated antibody. (B) The uptake of CFSE-labeled MSC-MVs was observed with flow cytometry. X-axis: the forward scatter corner signals. Y-axis: CFSE intensity. (C)The t-test showed that the PSR expression was significantly decreased by si-RNA (vs.CTR * P

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry, Fluorescence, Labeling

Suppression of MSC-MV incorporation into HUVECs by exogenous addition of Anx-V, anti-CD29 and anti-CD44 antibodies. (A) Flow cytometry was used to assess the CFSE intensity after the cells were cultured for 12 hours. CTR: HUVECs without MSC-MVs; MSC-MV: HUVECs with MSC-MVs only. X-axis: the forward scatter corner signals showing the cellular size. Y-axis: CFSE fluorescence intensity; (B) The percentages of the CFSE-positive cells are indicated. *P: vs .CTR
Figure Legend Snippet: Suppression of MSC-MV incorporation into HUVECs by exogenous addition of Anx-V, anti-CD29 and anti-CD44 antibodies. (A) Flow cytometry was used to assess the CFSE intensity after the cells were cultured for 12 hours. CTR: HUVECs without MSC-MVs; MSC-MV: HUVECs with MSC-MVs only. X-axis: the forward scatter corner signals showing the cellular size. Y-axis: CFSE fluorescence intensity; (B) The percentages of the CFSE-positive cells are indicated. *P: vs .CTR

Techniques Used: Flow Cytometry, Cytometry, Cell Culture, Fluorescence

MSC-MVs were engulfed by HUVECs dose- and time-dependent. (A) CFSE-labeled MSC-MVs were added to the culture medium of HUVECs at a dose of 10μg/ml and the cells were collected at different time-points. (B) Flow cytometry graphs analysis the mean fluorescent values of tripical experiments, error bars are +/- S.D.,*P
Figure Legend Snippet: MSC-MVs were engulfed by HUVECs dose- and time-dependent. (A) CFSE-labeled MSC-MVs were added to the culture medium of HUVECs at a dose of 10μg/ml and the cells were collected at different time-points. (B) Flow cytometry graphs analysis the mean fluorescent values of tripical experiments, error bars are +/- S.D.,*P

Techniques Used: Labeling, Flow Cytometry, Cytometry

Blockage of PSR suppresses the engulfment of MSC-MVs by HUVECs. (A) PSR expression on the HUVECs was detected with confocal microscopy analysis. PSR: green; DAPI: the nuclear (blue); Bar: 20μm. (B) HUVECs were pre-treated with anti-PSR antibody and cultured in the presence of DiI-labeled MSC-MVs. Twelve hours later, the cells were fixed and observed under a confocal microscope. CTR: HUVEC with DiI-MV; PSR-Antibody: HUVEC+PSR-Antibody with DiI-MV; Bar: 20μm. (C) Blockage of PSR with a specific antibody greatly decreased the internalization of MVs into HUVECs ( vs .CTR *** P
Figure Legend Snippet: Blockage of PSR suppresses the engulfment of MSC-MVs by HUVECs. (A) PSR expression on the HUVECs was detected with confocal microscopy analysis. PSR: green; DAPI: the nuclear (blue); Bar: 20μm. (B) HUVECs were pre-treated with anti-PSR antibody and cultured in the presence of DiI-labeled MSC-MVs. Twelve hours later, the cells were fixed and observed under a confocal microscope. CTR: HUVEC with DiI-MV; PSR-Antibody: HUVEC+PSR-Antibody with DiI-MV; Bar: 20μm. (C) Blockage of PSR with a specific antibody greatly decreased the internalization of MVs into HUVECs ( vs .CTR *** P

Techniques Used: Expressing, Confocal Microscopy, Cell Culture, Labeling, Microscopy

Blockage to PS on the MVs abrogates their ability to promote tube formation of HUVECs. (A) The vessel structure formation of HUVECs. CTR: Control, without MSC-MVs; MV: HUVEC+MSC-MVs; Anx-V-MV: HUVEC+Anx-V-MVs; (B) The mean number of 5 sites vessel structure (*P: vs .CTR
Figure Legend Snippet: Blockage to PS on the MVs abrogates their ability to promote tube formation of HUVECs. (A) The vessel structure formation of HUVECs. CTR: Control, without MSC-MVs; MV: HUVEC+MSC-MVs; Anx-V-MV: HUVEC+Anx-V-MVs; (B) The mean number of 5 sites vessel structure (*P: vs .CTR

Techniques Used:

Incorporation of MSC-MVs into HUVECs observed with confocal laser microscopy. (A) DiI-labeled MSC-MVs were added into the HUVEC culture. The cells were fixed and DAPI-stained, followed by confocal observation. Red: DiI-MVs; Blue: DAPI; Ordinary light: HUVEC; and Merge: the merged images of the three above. Bar: 10μm. (B) The mean fluorescence intensity of DiI in per field. The results are representative of three individual experiments.
Figure Legend Snippet: Incorporation of MSC-MVs into HUVECs observed with confocal laser microscopy. (A) DiI-labeled MSC-MVs were added into the HUVEC culture. The cells were fixed and DAPI-stained, followed by confocal observation. Red: DiI-MVs; Blue: DAPI; Ordinary light: HUVEC; and Merge: the merged images of the three above. Bar: 10μm. (B) The mean fluorescence intensity of DiI in per field. The results are representative of three individual experiments.

Techniques Used: Microscopy, Labeling, Staining, Fluorescence

Down-regulation of Anx-V in HUVECs has little effect on the MSC-MV internalization. (A) Anx-V siRNAs were transfected into HUVECs and the Anx-V expression was assessed by flow cytometry. The hollow diagrams represent the control and the solid ones represent the Anx-V-FITC fluorescence intensities of the indicated cells. (B) HUVECs, Anx-V siRNA-transfected HUVECs (siRNA) and control siRNA-transfected HUVECs (siRNA-CTR) were maintained in culture for 12 hours in the presence of CFSE-labeled MSC-MVs. The cells were harvested for flow cytometric analysis. (C) The t-test shows that the Anx-V expression was significantly decreased by si-RNA ( vs .CTR *P
Figure Legend Snippet: Down-regulation of Anx-V in HUVECs has little effect on the MSC-MV internalization. (A) Anx-V siRNAs were transfected into HUVECs and the Anx-V expression was assessed by flow cytometry. The hollow diagrams represent the control and the solid ones represent the Anx-V-FITC fluorescence intensities of the indicated cells. (B) HUVECs, Anx-V siRNA-transfected HUVECs (siRNA) and control siRNA-transfected HUVECs (siRNA-CTR) were maintained in culture for 12 hours in the presence of CFSE-labeled MSC-MVs. The cells were harvested for flow cytometric analysis. (C) The t-test shows that the Anx-V expression was significantly decreased by si-RNA ( vs .CTR *P

Techniques Used: Transfection, Expressing, Flow Cytometry, Cytometry, Fluorescence, Labeling

Identification of MSC-MVs with electron microscopy (A) and flow cytometry (B). (A) The bar represents 100nm. (B) MSC-MVs were conjugated with aldehyde/sulfate latex beads and reacted with fluorescein-labeled antibodies or Anx-V. The events were collected with a flow cytometer and the single beads (red) and the doublets of beads (green) were gated for further analysis. The percentages of the positivity in contrast to an isotype antibody are indicated. X-axis: forward scatter corner signals showing the size of the gated events. Beads: Beads were collected for the determination of the gates. Beads+MVs: MVs conjugated with beads were collected for further determination of the gates for analysis. CTR: MVs reacted with a PE-labeled isotype antibody. The results are representative of three individual experiments.
Figure Legend Snippet: Identification of MSC-MVs with electron microscopy (A) and flow cytometry (B). (A) The bar represents 100nm. (B) MSC-MVs were conjugated with aldehyde/sulfate latex beads and reacted with fluorescein-labeled antibodies or Anx-V. The events were collected with a flow cytometer and the single beads (red) and the doublets of beads (green) were gated for further analysis. The percentages of the positivity in contrast to an isotype antibody are indicated. X-axis: forward scatter corner signals showing the size of the gated events. Beads: Beads were collected for the determination of the gates. Beads+MVs: MVs conjugated with beads were collected for further determination of the gates for analysis. CTR: MVs reacted with a PE-labeled isotype antibody. The results are representative of three individual experiments.

Techniques Used: Electron Microscopy, Flow Cytometry, Cytometry, Labeling

11) Product Images from "Fat pad‐derived mesenchymal stem cells as a potential source for cell‐based adipose tissue repair strategies"

Article Title: Fat pad‐derived mesenchymal stem cells as a potential source for cell‐based adipose tissue repair strategies

Journal: Cell Proliferation

doi: 10.1111/j.1365-2184.2011.00804.x

), passage 10 (b) and passage 18 (c) fat pad‐derived MSCs using a panel of antibodies. Cell‐surface staining using FITC conjugated secondary antibody (green) and DAPI (blue) shows that cells stained strongly for CD13, CD29, CD44, CD90 and CD105, and poorly for LNGFR, STRO1, CD34 and CD56. Occasional cells stained for 3G5. No staining was observed for on IgG controls. The staining pattern was confirmed by flow cytometry, and shows increase in fluorescence (green) compared to autofluorescence (black) for CD13, CD29, CD44, CD90 and CD105.
Figure Legend Snippet: ), passage 10 (b) and passage 18 (c) fat pad‐derived MSCs using a panel of antibodies. Cell‐surface staining using FITC conjugated secondary antibody (green) and DAPI (blue) shows that cells stained strongly for CD13, CD29, CD44, CD90 and CD105, and poorly for LNGFR, STRO1, CD34 and CD56. Occasional cells stained for 3G5. No staining was observed for on IgG controls. The staining pattern was confirmed by flow cytometry, and shows increase in fluorescence (green) compared to autofluorescence (black) for CD13, CD29, CD44, CD90 and CD105.

Techniques Used: Derivative Assay, Staining, Flow Cytometry, Fluorescence

12) Product Images from "Exosomal microRNA-146a derived from mesenchymal stem cells increases the sensitivity of ovarian cancer cells to docetaxel and taxane via a LAMC2-mediated PI3K/Akt axis"

Article Title: Exosomal microRNA-146a derived from mesenchymal stem cells increases the sensitivity of ovarian cancer cells to docetaxel and taxane via a LAMC2-mediated PI3K/Akt axis

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2020.4634

Identification of hUCMSCs and their derived exosomes. (A) Expression of MSCs surface markers, including CD29, CD44, CD73, CD90, CD105, CD14, CD34, CD45 and HLA-DR, analyzed by flow cytometry. (B) hUCMSC morphology at passage 3 observed under a light microscope. Scale bar, 100 µ m. (C) Representative images of osteogenic differentiation of hUCMSCs using Alizarin Red staining. Scale bar, 100 µ m. (D) Representative images of adipogenic differentiation of hUCMSCs using Oil red O staining. Scale bar, 100 µ m. (E) Morphological analysis of hUCMSC by transmission electronic microscopy. Scale bar, 200 nm. (F) Detection of exosomal marker expression in hUCMSC-released exosomes by western blotting. (G) Internalization of PKH26-labeled exosomes by SKOV3 and A2780 cells was observed under a fluorescence microscope. Scale bar, 50 µ m. hUCMSCs, human umbilical cord mesenchymal stem cells.
Figure Legend Snippet: Identification of hUCMSCs and their derived exosomes. (A) Expression of MSCs surface markers, including CD29, CD44, CD73, CD90, CD105, CD14, CD34, CD45 and HLA-DR, analyzed by flow cytometry. (B) hUCMSC morphology at passage 3 observed under a light microscope. Scale bar, 100 µ m. (C) Representative images of osteogenic differentiation of hUCMSCs using Alizarin Red staining. Scale bar, 100 µ m. (D) Representative images of adipogenic differentiation of hUCMSCs using Oil red O staining. Scale bar, 100 µ m. (E) Morphological analysis of hUCMSC by transmission electronic microscopy. Scale bar, 200 nm. (F) Detection of exosomal marker expression in hUCMSC-released exosomes by western blotting. (G) Internalization of PKH26-labeled exosomes by SKOV3 and A2780 cells was observed under a fluorescence microscope. Scale bar, 50 µ m. hUCMSCs, human umbilical cord mesenchymal stem cells.

Techniques Used: Derivative Assay, Expressing, Flow Cytometry, Light Microscopy, Staining, Transmission Assay, Microscopy, Marker, Western Blot, Labeling, Fluorescence

13) Product Images from "Integrins Modulate T Cell Receptor Signaling by Constraining Actin Flow at the Immunological Synapse"

Article Title: Integrins Modulate T Cell Receptor Signaling by Constraining Actin Flow at the Immunological Synapse

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2018.00025

Focal adhesion proteins are recruited to sites of integrin engagement. (A) Primary human T cells were stimulated on coverslips patterned with ICAM-1 surrounded with OKT3 for 15 min, and labeled with m24 to detect the active, extended-open conformation of LFA-1, and Kim127 to detect the extended form of LFA-1. (B,C) Jurkat T cells were stimulated on VCAM-1 patterns surrounded with anti-CD3 for 15 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin, vinculin, and paxillin. (D,E) Primary human T cells were allowed to interact with VCAM-1 (D) or ICAM-1 (E) patterns surrounded with OKT3 for 17 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin and vinculin. Far right panels in (B–E) show cropped regions in which the four channels have been merged. (E) Scale bars = 10 µm.
Figure Legend Snippet: Focal adhesion proteins are recruited to sites of integrin engagement. (A) Primary human T cells were stimulated on coverslips patterned with ICAM-1 surrounded with OKT3 for 15 min, and labeled with m24 to detect the active, extended-open conformation of LFA-1, and Kim127 to detect the extended form of LFA-1. (B,C) Jurkat T cells were stimulated on VCAM-1 patterns surrounded with anti-CD3 for 15 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin, vinculin, and paxillin. (D,E) Primary human T cells were allowed to interact with VCAM-1 (D) or ICAM-1 (E) patterns surrounded with OKT3 for 17 min. Cells were then fixed and labeled with phalloidin to detect F-actin, and with antibodies to talin and vinculin. Far right panels in (B–E) show cropped regions in which the four channels have been merged. (E) Scale bars = 10 µm.

Techniques Used: Labeling

Integrin engagement slows actin flow. Jurkat T lymphoma cells and primary human CD4 + T cell blasts were allowed to interact with coverslips coated with anti-CD3 (OKT3) plus ICAM-1, VCAM-1, or both, and imaged for 4 min. (A) Still images of responding Jurkat cells stimulated on coverslips coated with anti-CD3 alone (left) or in the presence of VCAM-1 (right), together with corresponding kymographs of F-actin dynamics generated along the yellow lines. The white lines in the kymographs show example slopes used to calculate actin flow rates. (B) Kymographic analysis of F-actin features in Jurkat cells, showing the distribution of F-actin velocity across the immunological synapse (IS). The marked area displays the peripheral lamellipodial region (LP). (C,D) Surface expression of integrin LFA-1, as detected by CD11a staining and VLA-4, as detected by CD29 staining on Jurkat T cells (pink) and primary human CD4 + T cell blasts (blue). (E,F) Actin flow rates in the LP region for Jurkat cells (E) and primary human CD4 + T cell blasts (F) . Error bars show mean ± SD. * p
Figure Legend Snippet: Integrin engagement slows actin flow. Jurkat T lymphoma cells and primary human CD4 + T cell blasts were allowed to interact with coverslips coated with anti-CD3 (OKT3) plus ICAM-1, VCAM-1, or both, and imaged for 4 min. (A) Still images of responding Jurkat cells stimulated on coverslips coated with anti-CD3 alone (left) or in the presence of VCAM-1 (right), together with corresponding kymographs of F-actin dynamics generated along the yellow lines. The white lines in the kymographs show example slopes used to calculate actin flow rates. (B) Kymographic analysis of F-actin features in Jurkat cells, showing the distribution of F-actin velocity across the immunological synapse (IS). The marked area displays the peripheral lamellipodial region (LP). (C,D) Surface expression of integrin LFA-1, as detected by CD11a staining and VLA-4, as detected by CD29 staining on Jurkat T cells (pink) and primary human CD4 + T cell blasts (blue). (E,F) Actin flow rates in the LP region for Jurkat cells (E) and primary human CD4 + T cell blasts (F) . Error bars show mean ± SD. * p

Techniques Used: Flow Cytometry, Generated, Expressing, Staining

14) Product Images from "Do we really need to differentiate mesenchymal stem cells into insulin-producing cells for attenuation of the autoimmune responses in type 1 diabetes: immunoprophylactic effects of precursors to insulin-producing cells"

Article Title: Do we really need to differentiate mesenchymal stem cells into insulin-producing cells for attenuation of the autoimmune responses in type 1 diabetes: immunoprophylactic effects of precursors to insulin-producing cells

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0615-1

Representative FACS profile for phenotypic characterization of mesenchymal stem cells ( MSCs ). a Histograms showing percentage of MSCs positive for CD29 (99.8%), CD44 (72.4%), CD73 (19.4%), and SCA-1 (85.3%). b FACS histograms showing percentage of the hematopoietic markers CD45 (0.72%), CD11b (0.44%), and CD34 (13.3%). c Bar graph of four independent experiments showing percent mean ± SEM for both MSCs and hematopoietic stem cells markers: CD29 = 98.85 ± 0.33%, CD44 = 75.20 ± 8.60%, CD73 = 21.98 ± 1.81%, SCA-1 = 78.13 ± 4.64%, CD45 = 1.62 ± 0.44%, CD11b = 1.29 ± 0.54%, and CD34 = 27.40 ± 7.01% ( n = 4)
Figure Legend Snippet: Representative FACS profile for phenotypic characterization of mesenchymal stem cells ( MSCs ). a Histograms showing percentage of MSCs positive for CD29 (99.8%), CD44 (72.4%), CD73 (19.4%), and SCA-1 (85.3%). b FACS histograms showing percentage of the hematopoietic markers CD45 (0.72%), CD11b (0.44%), and CD34 (13.3%). c Bar graph of four independent experiments showing percent mean ± SEM for both MSCs and hematopoietic stem cells markers: CD29 = 98.85 ± 0.33%, CD44 = 75.20 ± 8.60%, CD73 = 21.98 ± 1.81%, SCA-1 = 78.13 ± 4.64%, CD45 = 1.62 ± 0.44%, CD11b = 1.29 ± 0.54%, and CD34 = 27.40 ± 7.01% ( n = 4)

Techniques Used: FACS

15) Product Images from "Differentiation of human umbilical cord mesenchymal stem cells into germ-like cells in mouse seminiferous tubules"

Article Title: Differentiation of human umbilical cord mesenchymal stem cells into germ-like cells in mouse seminiferous tubules

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2015.3528

Cellular characteristics of HUMSCs. (A) HUMSCs were observed to migrate out of the tissue fragment and exhibited a fibroblast-like morphology at passages (B) 1, (C) 3 and (D) 9. (E) Flow cytometric analysis of HUMSCs using antibodies against the human stem cell markers octamer-binding transcription factor 4, CD29, CD44 and CD59. PE- or FITC-labeled isotype antibodies were used as controls. Magnification, ×100. HUMSCs, human umbilical cord mesenchymal stem cells; PE, phycoerythrin; FITC, fluorescein isothiocyanate.
Figure Legend Snippet: Cellular characteristics of HUMSCs. (A) HUMSCs were observed to migrate out of the tissue fragment and exhibited a fibroblast-like morphology at passages (B) 1, (C) 3 and (D) 9. (E) Flow cytometric analysis of HUMSCs using antibodies against the human stem cell markers octamer-binding transcription factor 4, CD29, CD44 and CD59. PE- or FITC-labeled isotype antibodies were used as controls. Magnification, ×100. HUMSCs, human umbilical cord mesenchymal stem cells; PE, phycoerythrin; FITC, fluorescein isothiocyanate.

Techniques Used: Flow Cytometry, Binding Assay, Labeling

16) Product Images from "Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks"

Article Title: Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-10-98

Expression of cellular marker molecules and extracellular matrix (ECM) proteins by human umbilical cord artery derived cells (HUCAC). Using indirect immunofluorescence staining, highly positive signals (green) were detected for A ) collagen type I of fresh cultivated cells and B ) collagen type I of cryopreserved cells, E ) collagen type III of fresh cultivated cells and F ) collagen type III of cryopreserved cells. The presence of C ) collagen type I (green) and G ) collagen type III (green) was shown in native human umbilical cord artery walls, serving as a control. Immunohistochemical staining verified the presence of D ) collagen type I (red) and H ) collagen type III (red) in native human umbilical cord artery walls. Using flow cytometry analysis, cellular marker expression of short-term (group A, n = 4) and long-term (group B, n = 4) cryopreserved cells from primary cultures (passage 0) was studied directly after I ) thawing and J ) in passage 3 of recultivation. By comparison, non-cryopreserved fresh cells (n = 3) from I ) primary cultures and J ) passage 3 were analyzed in parallel as a control group Using indirect immunofluorescence staining, highly positive signals (green) were detected for all cellular markers tested such as K ) CD90 (green)/ alpha smooth muscle actin (ASMA) (red) of fresh cultivated cells and L ) CD90 (green)/ ASMA (red) of cryopreserved cells, N ) CD29 of fresh cultivated cells and O ) CD29 of cryopreserved cells, P ) CD105 of fresh cultivated cells and Q ) CD105 of cryopreserved cells. Cell nuclei staining is pictured in blue, present in A-H and K-Q. All studies of marker expression are exemplarily shown for cells of passage 3.
Figure Legend Snippet: Expression of cellular marker molecules and extracellular matrix (ECM) proteins by human umbilical cord artery derived cells (HUCAC). Using indirect immunofluorescence staining, highly positive signals (green) were detected for A ) collagen type I of fresh cultivated cells and B ) collagen type I of cryopreserved cells, E ) collagen type III of fresh cultivated cells and F ) collagen type III of cryopreserved cells. The presence of C ) collagen type I (green) and G ) collagen type III (green) was shown in native human umbilical cord artery walls, serving as a control. Immunohistochemical staining verified the presence of D ) collagen type I (red) and H ) collagen type III (red) in native human umbilical cord artery walls. Using flow cytometry analysis, cellular marker expression of short-term (group A, n = 4) and long-term (group B, n = 4) cryopreserved cells from primary cultures (passage 0) was studied directly after I ) thawing and J ) in passage 3 of recultivation. By comparison, non-cryopreserved fresh cells (n = 3) from I ) primary cultures and J ) passage 3 were analyzed in parallel as a control group Using indirect immunofluorescence staining, highly positive signals (green) were detected for all cellular markers tested such as K ) CD90 (green)/ alpha smooth muscle actin (ASMA) (red) of fresh cultivated cells and L ) CD90 (green)/ ASMA (red) of cryopreserved cells, N ) CD29 of fresh cultivated cells and O ) CD29 of cryopreserved cells, P ) CD105 of fresh cultivated cells and Q ) CD105 of cryopreserved cells. Cell nuclei staining is pictured in blue, present in A-H and K-Q. All studies of marker expression are exemplarily shown for cells of passage 3.

Techniques Used: Expressing, Marker, Derivative Assay, Immunofluorescence, Staining, Immunohistochemistry, Flow Cytometry, Cytometry

17) Product Images from "Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells"

Article Title: Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells

Journal: Scientific Reports

doi: 10.1038/srep17483

Characterization of DPCs: ( a ) After being separately cultured in osteogenic (Os) and adipogenic (Ad) media for 21 days, mineralized nodules were stained with alizarin red and oil red staining was used to assess the formation of oil droplets. DPCs which were cultured in neurogenic (Ne) media for 2 hours formed the axon-like structure. ( b ) Isolated DPCs were positive for Vimentin and negative for CK14 by immunocytochemistry; Ape1 was strongly expressed in DPCs, and were located in the nuclei at the same time; ( c ) Flow cytometric analysis revealed that cultured DPCs are positive for CD29 (99.8%), CD44 (99.9%), CD90 (99.9%), CD105 (99.3%) and CD166 (99.9%) but negative for CD14 (0.2%), CD31 (0.2%), CD34 (0.1%) and CD45 (0.0%). Mouse IgG isotype control antibodies conjugated to FITC, PE, or APC were used as negative controls. Scale bars: 50 μm.
Figure Legend Snippet: Characterization of DPCs: ( a ) After being separately cultured in osteogenic (Os) and adipogenic (Ad) media for 21 days, mineralized nodules were stained with alizarin red and oil red staining was used to assess the formation of oil droplets. DPCs which were cultured in neurogenic (Ne) media for 2 hours formed the axon-like structure. ( b ) Isolated DPCs were positive for Vimentin and negative for CK14 by immunocytochemistry; Ape1 was strongly expressed in DPCs, and were located in the nuclei at the same time; ( c ) Flow cytometric analysis revealed that cultured DPCs are positive for CD29 (99.8%), CD44 (99.9%), CD90 (99.9%), CD105 (99.3%) and CD166 (99.9%) but negative for CD14 (0.2%), CD31 (0.2%), CD34 (0.1%) and CD45 (0.0%). Mouse IgG isotype control antibodies conjugated to FITC, PE, or APC were used as negative controls. Scale bars: 50 μm.

Techniques Used: Cell Culture, Staining, Isolation, Immunocytochemistry, Flow Cytometry

18) Product Images from "The effects of bone marrow-derived mesenchymal stem cells on ovalbumin-induced allergic asthma and cytokine responses in mice"

Article Title: The effects of bone marrow-derived mesenchymal stem cells on ovalbumin-induced allergic asthma and cytokine responses in mice

Journal: Iranian Journal of Basic Medical Sciences

doi: 10.22038/IJBMS.2018.26898.6575

Flow cytometric analysis of cell surface markers of mice bone marrow derived MSCs. The respective isotype control is shown as white. CD29 (92.38%), CD44 (79.66), CD90 (96.27%), Stem cell antigen (Sca)-1 (82.12%), CD34 (0.8%), CD45 (0.5%), CD14 (0.6%), and CD11b (0.5%)
Figure Legend Snippet: Flow cytometric analysis of cell surface markers of mice bone marrow derived MSCs. The respective isotype control is shown as white. CD29 (92.38%), CD44 (79.66), CD90 (96.27%), Stem cell antigen (Sca)-1 (82.12%), CD34 (0.8%), CD45 (0.5%), CD14 (0.6%), and CD11b (0.5%)

Techniques Used: Flow Cytometry, Mouse Assay, Derivative Assay

19) Product Images from "Combined platelet-rich plasma and lipofilling treatment provides great improvement in facial skin-induced lesion regeneration for scleroderma patients"

Article Title: Combined platelet-rich plasma and lipofilling treatment provides great improvement in facial skin-induced lesion regeneration for scleroderma patients

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0690-3

SSc adipose samples show peculiar features and different tissue morphology. a Representative H E staining on paraffin-embedded sections of freshly isolated adipose tissue from healthy donors and SSc patients. Arrows indicate MSCs (black) and blood vessels (red). b CD271 positivity in freshly isolated SVF and long-term propagated AD-MSCs from healthy donors and SSc patients, performed by flow cytometry. Data are mean ± SD of three independent experiments. c Flow cytometry analysis of cell positivity percentage for CD271, CD44, CD29, CD9, CD90, and CD73, performed on AD-MSCs of healthy subjects (blue) and SSc patients (red). Percentage values in b and c represent the percentage of parent population. Bars represent mean ± SD of three independent experiments. * p
Figure Legend Snippet: SSc adipose samples show peculiar features and different tissue morphology. a Representative H E staining on paraffin-embedded sections of freshly isolated adipose tissue from healthy donors and SSc patients. Arrows indicate MSCs (black) and blood vessels (red). b CD271 positivity in freshly isolated SVF and long-term propagated AD-MSCs from healthy donors and SSc patients, performed by flow cytometry. Data are mean ± SD of three independent experiments. c Flow cytometry analysis of cell positivity percentage for CD271, CD44, CD29, CD9, CD90, and CD73, performed on AD-MSCs of healthy subjects (blue) and SSc patients (red). Percentage values in b and c represent the percentage of parent population. Bars represent mean ± SD of three independent experiments. * p

Techniques Used: Staining, Isolation, Flow Cytometry, Cytometry

20) Product Images from "miR-138-mediated Regulation of Kindlin-2 Expression Modulates Sensitivity to Chemotherapeutics"

Article Title: miR-138-mediated Regulation of Kindlin-2 Expression Modulates Sensitivity to Chemotherapeutics

Journal: Molecular cancer research : MCR

doi: 10.1158/1541-7786.MCR-15-0299

Combination of Kindlin-2 knockdown and docetaxel treatment has a dramatic inhibitory effect on integrin β1-mediated cell spreading (A, B C) Representative histograms using flow cytometry of PC3 cells with the indicated transfections and staining with PE-conjugated anti CD29 antibody for cell surface β1 integrin (A B) and with FITC-conjugated anti CD61 antibody for β3 integrin (C). (D) Western blots with anti-K2 antibody of cell lysates from PC3 cells with the indicated transfections. β-Actin was used as a loading control. (E) Representative micrographs of Alexa 568 phalloidin-stained PC3 cells that were transfected with either a non-targeting siRNA (NT) or Kindlin-2 siRNA and seeded on uncoated coverslips (No Ligand) or on coverslips coated with fibronectin (20μg/ml) for 2 hrs in the presence or absence of docetaxel (Doc). (F) Quantification of PC3 cell spreading under the indicated treatments. Data are the fold-change in cell spreading normalized to the values for untreated and non-targeting siRNA transfected cells. Data are representative of 3 independent experiments (*, p
Figure Legend Snippet: Combination of Kindlin-2 knockdown and docetaxel treatment has a dramatic inhibitory effect on integrin β1-mediated cell spreading (A, B C) Representative histograms using flow cytometry of PC3 cells with the indicated transfections and staining with PE-conjugated anti CD29 antibody for cell surface β1 integrin (A B) and with FITC-conjugated anti CD61 antibody for β3 integrin (C). (D) Western blots with anti-K2 antibody of cell lysates from PC3 cells with the indicated transfections. β-Actin was used as a loading control. (E) Representative micrographs of Alexa 568 phalloidin-stained PC3 cells that were transfected with either a non-targeting siRNA (NT) or Kindlin-2 siRNA and seeded on uncoated coverslips (No Ligand) or on coverslips coated with fibronectin (20μg/ml) for 2 hrs in the presence or absence of docetaxel (Doc). (F) Quantification of PC3 cell spreading under the indicated treatments. Data are the fold-change in cell spreading normalized to the values for untreated and non-targeting siRNA transfected cells. Data are representative of 3 independent experiments (*, p

Techniques Used: Flow Cytometry, Cytometry, Transfection, Staining, Western Blot

21) Product Images from "Intercellular Adhesion Molecule-1 (ICAM-1) and ICAM-2 Differentially Contribute to Peripheral Activation and CNS Entry of Autoaggressive Th1 and Th17 Cells in Experimental Autoimmune Encephalomyelitis"

Article Title: Intercellular Adhesion Molecule-1 (ICAM-1) and ICAM-2 Differentially Contribute to Peripheral Activation and CNS Entry of Autoaggressive Th1 and Th17 Cells in Experimental Autoimmune Encephalomyelitis

Journal: Frontiers in Immunology

doi: 10.3389/fimmu.2019.03056

Lack of ICAM-1 and ICAM-2 impairs firm adhesion and crawling of in vitro polarized Th1 and Th17 CD4 + cells with the inflamed BBB in vivo . (A) Representative frames of epifluorescence-IVM imaging of Th1 or Th17 cells adhering in inflamed cervical spinal cord microvessels of WT or ICAM-1/-2 −/− C57BL/6J mice with EAE 60 min post-infusion. White arrows indicate arrested Th1 or Th17 cells in cervical spinal cord microvasculature. (B) Number of adhered Th1 and Th17 cells in inflamed cervical spinal cord microvessels of WT and ICAM-1/-2 −/− C57BL/6J mice with EAE counted at 10, 30, and 60 min after infusion of Th1 and Th17 cells. Data are pooled from imaging of 4 EAE mice per each condition and are shown as mean ± SEM. (C,D) Movement of fluorescently labeled Th1 and Th17 CD4 + cells on the inflamed BBB of WT (C) or ICAM-1/-2 −/− (D) EAE mice is shown at 0, 4, 6, 8, and 11 min of recording. Vascular endothelial cells were labeled by infusion of fluorophore-conjugated rat-anti-mouse endoglin antibody (CD105: 2 μg/mouse). Time shown in minutes and seconds. Scale bar: 40 μm. (C) Blue arrows indicate CellTracker Blue (CMAC) labeled Th1 cells and green arrows show CellTracker Green (CMFDA) labeled Th17 cells interacting with spinal cord microvessels of WT C57BL/6J mice with EAE. Both Th1 and Th17 cells crawled with and against the direction of blood flow (yellow arrow). (D) Green arrow shows CMFDA labeled Th1 cells; blue arrow shows CMAC labeled Th17 cells interacting with spinal cord microvessels of ICAM-1/-2 −/− C57BL/6J mice during EAE. Adhering Th1 and Th17 cells on the inflamed BBB either detached (yellow arrow) and recirculated or failed to crawl in inflamed ICAM-1/-2 −/− microvessels during EAE. (E,F) Intraluminal crawling of fluorescently labeled Th1 or Th17 cells on endothelial cells of inflamed spinal cord microvessels were imaged using time-lapse 2P-IVM. Each data point represents one individual CD4 + Th1 or Th17 cells track. Speed (E) and displacement rate (F) of individual interacting Th1 and Th17 CD4 + cells with inflamed BBB of WT and ICAM-1/-2 −/− C57BL/6J mice suffering from EAE were calculated using Volocity software. Values are pooled from imaging of 4 EAE mice per each condition and are shown as ± SEM. Data were analyzed using Mann-Whitney-Test to compare each two groups * p
Figure Legend Snippet: Lack of ICAM-1 and ICAM-2 impairs firm adhesion and crawling of in vitro polarized Th1 and Th17 CD4 + cells with the inflamed BBB in vivo . (A) Representative frames of epifluorescence-IVM imaging of Th1 or Th17 cells adhering in inflamed cervical spinal cord microvessels of WT or ICAM-1/-2 −/− C57BL/6J mice with EAE 60 min post-infusion. White arrows indicate arrested Th1 or Th17 cells in cervical spinal cord microvasculature. (B) Number of adhered Th1 and Th17 cells in inflamed cervical spinal cord microvessels of WT and ICAM-1/-2 −/− C57BL/6J mice with EAE counted at 10, 30, and 60 min after infusion of Th1 and Th17 cells. Data are pooled from imaging of 4 EAE mice per each condition and are shown as mean ± SEM. (C,D) Movement of fluorescently labeled Th1 and Th17 CD4 + cells on the inflamed BBB of WT (C) or ICAM-1/-2 −/− (D) EAE mice is shown at 0, 4, 6, 8, and 11 min of recording. Vascular endothelial cells were labeled by infusion of fluorophore-conjugated rat-anti-mouse endoglin antibody (CD105: 2 μg/mouse). Time shown in minutes and seconds. Scale bar: 40 μm. (C) Blue arrows indicate CellTracker Blue (CMAC) labeled Th1 cells and green arrows show CellTracker Green (CMFDA) labeled Th17 cells interacting with spinal cord microvessels of WT C57BL/6J mice with EAE. Both Th1 and Th17 cells crawled with and against the direction of blood flow (yellow arrow). (D) Green arrow shows CMFDA labeled Th1 cells; blue arrow shows CMAC labeled Th17 cells interacting with spinal cord microvessels of ICAM-1/-2 −/− C57BL/6J mice during EAE. Adhering Th1 and Th17 cells on the inflamed BBB either detached (yellow arrow) and recirculated or failed to crawl in inflamed ICAM-1/-2 −/− microvessels during EAE. (E,F) Intraluminal crawling of fluorescently labeled Th1 or Th17 cells on endothelial cells of inflamed spinal cord microvessels were imaged using time-lapse 2P-IVM. Each data point represents one individual CD4 + Th1 or Th17 cells track. Speed (E) and displacement rate (F) of individual interacting Th1 and Th17 CD4 + cells with inflamed BBB of WT and ICAM-1/-2 −/− C57BL/6J mice suffering from EAE were calculated using Volocity software. Values are pooled from imaging of 4 EAE mice per each condition and are shown as ± SEM. Data were analyzed using Mann-Whitney-Test to compare each two groups * p

Techniques Used: In Vitro, In Vivo, Imaging, Mouse Assay, Labeling, Software, MANN-WHITNEY

Important role of ICAM-1 and ICAM-2 on DCs in MOG aa35−55 -specific T-cell proliferation and activation in peripheral lymph nodes of C57BL/6J mice. (A–I) Proliferation of e670-labeled 2D2-GFP CD4 + T cells 48 and 72 h after transfer into recipient mice prior injected with MOG aa35−55 -pulsed WT or ICAM-1/-2 −/− DCs. DCs were pulsed with 2, 100 μg/ml, or no (BASAL) MOG aa35−55 peptide. (A) Representative FACS dot plots showing percentage of divided, undivided and each individual generation of divided 2D2-GFP CD4 + T cells (generations 1–5) 48 h after adoptive transfer into recipient mice. Gates and the corresponding percentages of undivided cells shown in black, total divided cells in red, generation 1 of divided cells in orange, generation 2 in brown, generation 3 in green, generation 4 in purple, and generation 5 in blue. (B,C) Quantification of divided (B) or undivided (C) e670-labeled 2D2 GFP CD4 + T cells percentage after in vivo interaction with WT or ICAM-1/-2 −/− DCs pulsed with 2, 100 μg/ml, or no (BASAL) MOG aa35−55 peptide. Cell divisions were monitored via dilution of the e670 fluorescent dye, 48 and 72 h after transfer of e670-labeled 2D2 GFP CD4 + T cells into recipient mice. Each dot represents pLN T cells pooled from one mouse. Data pooled from two individual experiments with a total of 7–8 mice at each time point and 3–4 mice at BASAL level. (D–I) Cell surface expression of CD69 and CD25 within all (D,G) , divided (E,H) , and undivided (F,I) 2D2 GFP CD4 + T cell-subsets shown as percentage after in vivo interaction with WT or ICAM-1/-2 −/− DCs pulsed with 2, 100 μg/ml, or no (BASAL) MOG aa35−55 peptide. Each dot in the plots represents pLN T cells pooled from one mouse. Results are representative data from two separate experiments with 5 mice in each group (except BASAL level with 3–4 mice) and shown as mean. Data were analyzed using repeated measure ANOVA with Bonferroni post-test. * p
Figure Legend Snippet: Important role of ICAM-1 and ICAM-2 on DCs in MOG aa35−55 -specific T-cell proliferation and activation in peripheral lymph nodes of C57BL/6J mice. (A–I) Proliferation of e670-labeled 2D2-GFP CD4 + T cells 48 and 72 h after transfer into recipient mice prior injected with MOG aa35−55 -pulsed WT or ICAM-1/-2 −/− DCs. DCs were pulsed with 2, 100 μg/ml, or no (BASAL) MOG aa35−55 peptide. (A) Representative FACS dot plots showing percentage of divided, undivided and each individual generation of divided 2D2-GFP CD4 + T cells (generations 1–5) 48 h after adoptive transfer into recipient mice. Gates and the corresponding percentages of undivided cells shown in black, total divided cells in red, generation 1 of divided cells in orange, generation 2 in brown, generation 3 in green, generation 4 in purple, and generation 5 in blue. (B,C) Quantification of divided (B) or undivided (C) e670-labeled 2D2 GFP CD4 + T cells percentage after in vivo interaction with WT or ICAM-1/-2 −/− DCs pulsed with 2, 100 μg/ml, or no (BASAL) MOG aa35−55 peptide. Cell divisions were monitored via dilution of the e670 fluorescent dye, 48 and 72 h after transfer of e670-labeled 2D2 GFP CD4 + T cells into recipient mice. Each dot represents pLN T cells pooled from one mouse. Data pooled from two individual experiments with a total of 7–8 mice at each time point and 3–4 mice at BASAL level. (D–I) Cell surface expression of CD69 and CD25 within all (D,G) , divided (E,H) , and undivided (F,I) 2D2 GFP CD4 + T cell-subsets shown as percentage after in vivo interaction with WT or ICAM-1/-2 −/− DCs pulsed with 2, 100 μg/ml, or no (BASAL) MOG aa35−55 peptide. Each dot in the plots represents pLN T cells pooled from one mouse. Results are representative data from two separate experiments with 5 mice in each group (except BASAL level with 3–4 mice) and shown as mean. Data were analyzed using repeated measure ANOVA with Bonferroni post-test. * p

Techniques Used: Activation Assay, Mouse Assay, Labeling, Injection, FACS, Adoptive Transfer Assay, In Vivo, Expressing

Reduced in vitro MOG aa35−55 -specific CD4 + T-cell proliferation in the absence of ICAM-1 but not ICAM-2 on DCs. (A–F) Purified CD4 + T cells (TC) were co-cultured with irradiated BM-derived DCs (DC) with a ratio of 1:10 DCs:TCs. Different concentrations of MOG aa35−55 peptide were added to the co-cultures and incubated for 72 h before pulsing with [ 3 H]-thymidine. 3 H-thymidine incorporation into TCs shown as counts per minute (CPM)/well. (A–C) TCs from 2D2 C57BL/6J mice co-cultured with irradiated DCs isolated from WT, ICAM-1 −/− (A) , ICAM-2 −/− (B) , or ICAM-1/-2 −/− (C) C57BL/6J mice. (D–F) TCs from 2D2, 2D2 ICAM-1 −/− (D) , 2D2 ICAM-2 −/− (E) or 2D2 ICAM-1/-2 −/− (F) C57BL/6J mice co-cultured with irradiated DCs from WT C57BL/6J mice. Results show mean ± SEM after subtraction of background proliferation determined in the absence of MOG aa35−55 peptide. Data are representative of 3-4 individual experiments per condition. Statistical differences between the groups calculated with paired Student's t -test. * p
Figure Legend Snippet: Reduced in vitro MOG aa35−55 -specific CD4 + T-cell proliferation in the absence of ICAM-1 but not ICAM-2 on DCs. (A–F) Purified CD4 + T cells (TC) were co-cultured with irradiated BM-derived DCs (DC) with a ratio of 1:10 DCs:TCs. Different concentrations of MOG aa35−55 peptide were added to the co-cultures and incubated for 72 h before pulsing with [ 3 H]-thymidine. 3 H-thymidine incorporation into TCs shown as counts per minute (CPM)/well. (A–C) TCs from 2D2 C57BL/6J mice co-cultured with irradiated DCs isolated from WT, ICAM-1 −/− (A) , ICAM-2 −/− (B) , or ICAM-1/-2 −/− (C) C57BL/6J mice. (D–F) TCs from 2D2, 2D2 ICAM-1 −/− (D) , 2D2 ICAM-2 −/− (E) or 2D2 ICAM-1/-2 −/− (F) C57BL/6J mice co-cultured with irradiated DCs from WT C57BL/6J mice. Results show mean ± SEM after subtraction of background proliferation determined in the absence of MOG aa35−55 peptide. Data are representative of 3-4 individual experiments per condition. Statistical differences between the groups calculated with paired Student's t -test. * p

Techniques Used: In Vitro, Purification, Cell Culture, Irradiation, Derivative Assay, Incubation, Mouse Assay, Isolation

Absence of ICAM-1 and ICAM-2 affects the interaction of in vitro polarized Th1 and Th17 CD4 + cells with an in vitro model of the BBB. (A) Mean number of arrested CD4 + Th1 (red) and Th17 (green) cells per FOV (872 × 654 μm) on WT (filled bars) and ICAM-1/-2 −/− (striped bars) non-stimulated (control), TNFα or IL-1β-stimulated pMBMECs. Each dot represents the number of arrested T cells in one experiment, for a total of four independent experiments. Data shown as mean ± SD. (B,C) Post-arrest dynamic behavior during 30 min of recording time of in vitro polarized CD4 + Th1 and Th17 cells on TNFα (B) or IL-1β (C) stimulated pMBMECs. Presence or absence of complete or partial diapedesis is indicated for each migratory behavioral category, as indicated. The behavioral categories are presented as percentage of categorized CD4 + Th1 and Th17 cells for each condition of pMBMECs. Data shown as mean ± SD from four independent experiments. (D) Crawling speed in μm/min of CD4 + Th1 and Th17 cells on WT (squares) and ICAM-1/-2 −/− (triangles) control, TNFα or IL-1β-stimulated pMBMECs. Each point represents one crawling track of one CD4 + Th1 or Th17 cell. Values shown as mean (Th1 or Th17 crawling tracks on WT pMBMECs ≥50; Th1 or Th17 crawling tracks on ICAM-1/-2 −/− pMBMECs ≤ 37) and are pooled from four individual experiments. Data were analyzed using repeated measure ANOVA with Tukey post-test * p
Figure Legend Snippet: Absence of ICAM-1 and ICAM-2 affects the interaction of in vitro polarized Th1 and Th17 CD4 + cells with an in vitro model of the BBB. (A) Mean number of arrested CD4 + Th1 (red) and Th17 (green) cells per FOV (872 × 654 μm) on WT (filled bars) and ICAM-1/-2 −/− (striped bars) non-stimulated (control), TNFα or IL-1β-stimulated pMBMECs. Each dot represents the number of arrested T cells in one experiment, for a total of four independent experiments. Data shown as mean ± SD. (B,C) Post-arrest dynamic behavior during 30 min of recording time of in vitro polarized CD4 + Th1 and Th17 cells on TNFα (B) or IL-1β (C) stimulated pMBMECs. Presence or absence of complete or partial diapedesis is indicated for each migratory behavioral category, as indicated. The behavioral categories are presented as percentage of categorized CD4 + Th1 and Th17 cells for each condition of pMBMECs. Data shown as mean ± SD from four independent experiments. (D) Crawling speed in μm/min of CD4 + Th1 and Th17 cells on WT (squares) and ICAM-1/-2 −/− (triangles) control, TNFα or IL-1β-stimulated pMBMECs. Each point represents one crawling track of one CD4 + Th1 or Th17 cell. Values shown as mean (Th1 or Th17 crawling tracks on WT pMBMECs ≥50; Th1 or Th17 crawling tracks on ICAM-1/-2 −/− pMBMECs ≤ 37) and are pooled from four individual experiments. Data were analyzed using repeated measure ANOVA with Tukey post-test * p

Techniques Used: In Vitro

ICAM-1/-2 −/− DCs display increased migration speed and shorter interaction times with 2D2 CD4 + T cells in popliteal LN in vivo . (A–D) WT and ICAM-1/-2 −/− DCs pulsed with 100 μg/ml MOG aa35−55 peptide were subcutaneously injected into WT C57BL/6J mice and allowed to home for 18 h to the dLN. Naïve 2D2-GFP + CD4 + T cells were adoptively transferred into recipient mice and their interaction with DCs in the popliteal LN were imaged starting within 10–15 min after injection. (A,B) Representative 2P-IVM images showing the interaction of WT and ICAM-1/-2 −/− DCs with 2D2-GFP CD4 + T cells. (A) Arrows indicate a WT DC (blue) and an ICAM-1/-2 −/− DC (red) interacting with 2D2-GFP CD4 + T cells during the entire time of imaging (20 min). Arrowheads show a transient interaction of a WT DC (blue) and an ICAM-1/-2 −/− DC (red) with 2D2-GFP CD4 + T cells during the 20 min recording. Time shown in minutes and seconds. Scale bar, 10 μm. (B) Arrow points to a 2D2 GFP CD4 + T cell positioned between one WT DC (blue) and one ICAM-1/-2 −/− DC (red) after migration across the HEV and subsequently interacting preferentially with the WT DC. High endothelial venule, labeled with Alexa Fluor 633–conjugated MECA-79 on the top right corner. Time shown in minutes and seconds. Scale bar, 10 μm. (C) Graphs show speed of individual WT and ICAM-1/-2 −/− DCs within the PLN and relative frequency of the speeds of the DCs as monitored by 2P-IVM imaging. (D) Interaction times between 2D2-GFP CD4 + T cells and WT or ICAM-1/-2 −/− DCs were monitored. Each dot represents one interaction (C) or one track (D) quantified over the entire 20 min of recorded movies using Volocity software. Data in (C) and (D) are pooled from two independent experiments and shown as median. Data were analyzed by unpaired Student's t -test. ** p
Figure Legend Snippet: ICAM-1/-2 −/− DCs display increased migration speed and shorter interaction times with 2D2 CD4 + T cells in popliteal LN in vivo . (A–D) WT and ICAM-1/-2 −/− DCs pulsed with 100 μg/ml MOG aa35−55 peptide were subcutaneously injected into WT C57BL/6J mice and allowed to home for 18 h to the dLN. Naïve 2D2-GFP + CD4 + T cells were adoptively transferred into recipient mice and their interaction with DCs in the popliteal LN were imaged starting within 10–15 min after injection. (A,B) Representative 2P-IVM images showing the interaction of WT and ICAM-1/-2 −/− DCs with 2D2-GFP CD4 + T cells. (A) Arrows indicate a WT DC (blue) and an ICAM-1/-2 −/− DC (red) interacting with 2D2-GFP CD4 + T cells during the entire time of imaging (20 min). Arrowheads show a transient interaction of a WT DC (blue) and an ICAM-1/-2 −/− DC (red) with 2D2-GFP CD4 + T cells during the 20 min recording. Time shown in minutes and seconds. Scale bar, 10 μm. (B) Arrow points to a 2D2 GFP CD4 + T cell positioned between one WT DC (blue) and one ICAM-1/-2 −/− DC (red) after migration across the HEV and subsequently interacting preferentially with the WT DC. High endothelial venule, labeled with Alexa Fluor 633–conjugated MECA-79 on the top right corner. Time shown in minutes and seconds. Scale bar, 10 μm. (C) Graphs show speed of individual WT and ICAM-1/-2 −/− DCs within the PLN and relative frequency of the speeds of the DCs as monitored by 2P-IVM imaging. (D) Interaction times between 2D2-GFP CD4 + T cells and WT or ICAM-1/-2 −/− DCs were monitored. Each dot represents one interaction (C) or one track (D) quantified over the entire 20 min of recorded movies using Volocity software. Data in (C) and (D) are pooled from two independent experiments and shown as median. Data were analyzed by unpaired Student's t -test. ** p

Techniques Used: Migration, In Vivo, Injection, Mouse Assay, Imaging, Labeling, Software

22) Product Images from "Enhanced chondrogenesis of bone marrow-derived stem cells by using a combinatory cell therapy strategy with BMP-2/TGF-β1, hypoxia, and COL1A1/HtrA1 siRNAs"

Article Title: Enhanced chondrogenesis of bone marrow-derived stem cells by using a combinatory cell therapy strategy with BMP-2/TGF-β1, hypoxia, and COL1A1/HtrA1 siRNAs

Journal: Scientific Reports

doi: 10.1038/s41598-017-03579-y

Immunophenotype of hBM-MSCs. The expression of CD29, CD44, CD73, CD90, CD105, CD146, CD14, CD34, CD45, CD64, and HLA-DR was analyzed by flow cytometry in hBM-MSC preparations from eight donors between passage 3 and 7 (n = 8). The graphs show the percentage of positive cells relative to an isotype control depending on the strain and passage. The means are represented by a bar for each passage.
Figure Legend Snippet: Immunophenotype of hBM-MSCs. The expression of CD29, CD44, CD73, CD90, CD105, CD146, CD14, CD34, CD45, CD64, and HLA-DR was analyzed by flow cytometry in hBM-MSC preparations from eight donors between passage 3 and 7 (n = 8). The graphs show the percentage of positive cells relative to an isotype control depending on the strain and passage. The means are represented by a bar for each passage.

Techniques Used: Expressing, Flow Cytometry, Cytometry

23) Product Images from "Intratracheal administration of adipose derived mesenchymal stem cells alleviates chronic asthma in a mouse model"

Article Title: Intratracheal administration of adipose derived mesenchymal stem cells alleviates chronic asthma in a mouse model

Journal: BMC Pulmonary Medicine

doi: 10.1186/s12890-018-0701-x

The identification of adipose-derived mesenchymal stem cells of mice (mASCs). a The morphology of mASCs under A microscope (400X); b The expression of CD90, CD44, CD29, CD34 and CD45 on the surface of mASCs by flow cytometry
Figure Legend Snippet: The identification of adipose-derived mesenchymal stem cells of mice (mASCs). a The morphology of mASCs under A microscope (400X); b The expression of CD90, CD44, CD29, CD34 and CD45 on the surface of mASCs by flow cytometry

Techniques Used: Derivative Assay, Mouse Assay, Microscopy, Expressing, Flow Cytometry, Cytometry

24) Product Images from "The Effect of Conditioned Media of Adipose-Derived Stem Cells on Wound Healing after Ablative Fractional Carbon Dioxide Laser Resurfacing"

Article Title: The Effect of Conditioned Media of Adipose-Derived Stem Cells on Wound Healing after Ablative Fractional Carbon Dioxide Laser Resurfacing

Journal: BioMed Research International

doi: 10.1155/2013/519126

Characterization of adipose stem cell. The primary adipose stem cells exhibited a typical fibroblast-like morphology (a1) and were harvested at passage 3 (a2) for flow cytometry analysis. The results confirmed that the cells express CD29 (99.96%, b1), CD90 (47.1%, b4), CD34 (0.87%, b2), and CD71 (0.94%, b3). PE (0.56%, b5) and FITC (1.09%, b6) were performed as negative control. Oil Red O staining of lipid after differentiation for 28 days (before staining, c1; after staining, c2) and Alizarin Red staining of calcium after differentiation (before staining, c3; after staining, c4).
Figure Legend Snippet: Characterization of adipose stem cell. The primary adipose stem cells exhibited a typical fibroblast-like morphology (a1) and were harvested at passage 3 (a2) for flow cytometry analysis. The results confirmed that the cells express CD29 (99.96%, b1), CD90 (47.1%, b4), CD34 (0.87%, b2), and CD71 (0.94%, b3). PE (0.56%, b5) and FITC (1.09%, b6) were performed as negative control. Oil Red O staining of lipid after differentiation for 28 days (before staining, c1; after staining, c2) and Alizarin Red staining of calcium after differentiation (before staining, c3; after staining, c4).

Techniques Used: Flow Cytometry, Cytometry, Negative Control, Staining

25) Product Images from "Hyperhomocysteinemia suppresses bone marrow CD34+/VEGF receptor 2+ cells and inhibits progenitor cell mobilization and homing to injured vasculature—a role of β1-integrin in progenitor cell migration and adhesion"

Article Title: Hyperhomocysteinemia suppresses bone marrow CD34+/VEGF receptor 2+ cells and inhibits progenitor cell mobilization and homing to injured vasculature—a role of β1-integrin in progenitor cell migration and adhesion

Journal: The FASEB Journal

doi: 10.1096/fj.14-267989

Hcy inhibits β1-integrin expression/activity in human CD34 + ECFCs. Confluent human CD34 + ECFCs treated with l -Hcy (250 µM) for 48 h were examined for integrin expression and activity by flow cytometry. Cells were harvested and incubated
Figure Legend Snippet: Hcy inhibits β1-integrin expression/activity in human CD34 + ECFCs. Confluent human CD34 + ECFCs treated with l -Hcy (250 µM) for 48 h were examined for integrin expression and activity by flow cytometry. Cells were harvested and incubated

Techniques Used: Expressing, Activity Assay, Flow Cytometry, Cytometry, Incubation

26) Product Images from "A Therapeutic Strategy for Spinal Cord Defect: Human Dental Follicle Cells Combined with Aligned PCL/PLGA Electrospun Material"

Article Title: A Therapeutic Strategy for Spinal Cord Defect: Human Dental Follicle Cells Combined with Aligned PCL/PLGA Electrospun Material

Journal: BioMed Research International

doi: 10.1155/2015/197183

Flow cytometric analyses for immunophenotypic characteristics of hDFCs. hDFCs were positive for CD29, CD44, CD90, CD105, CD146, and CD166 and negative for CD24, CD34, and CD45. Green color stands for being labeled by FITC and red color stands for being labeled by PE.
Figure Legend Snippet: Flow cytometric analyses for immunophenotypic characteristics of hDFCs. hDFCs were positive for CD29, CD44, CD90, CD105, CD146, and CD166 and negative for CD24, CD34, and CD45. Green color stands for being labeled by FITC and red color stands for being labeled by PE.

Techniques Used: Flow Cytometry, Labeling

27) Product Images from "Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue"

Article Title: Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue

Journal: Molecular Metabolism

doi: 10.1016/j.molmet.2015.07.010

Id3 promotes HFD-induced MCP-1 in VAT . (A, B, G) SVF was isolated from epididymal VAT of 8 to 10 week old Id3 +/+ and Id3 −/− mice fed 1 week of either chow or HFD. n = 7–10. Flow quantitation of MCP-1 hi cells (A) and MCP-1 mid cells (B) per mouse (paired eVAT depots). (C–E) Epididymal VAT and subcutaneous adipose tissue were harvested from 8 to 10 week old Id3 +/+ and Id3 −/− mice fed 4 weeks of either chow or HFD. n = 5–6. (C, D) MCP-1 mRNA levels in epididymal (C) and subcutaneous (D) adipose, represented as fold increase over Id3 +/+ chow. (E) MCP-1 levels as measured by ELISA in the supernatant of epididymal VAT, cultured for 24 h. MCP-1 secretion was normalized per mouse (paired eVAT depots). (F) Weights of epididymal VAT from 8 to 10 week old Id3 +/+ and Id3 −/− mice fed chow-diet or 1 or 4 weeks of HFD. (G) Correlation of quantified MCP-1 hi cells with epididymal VAT weight in 1 week HFD-fed Id3 +/+ and Id3 −/− mice. (H) MCP-1 promoter activity in OP-9 cells, transfected with plasmid encoding ID3 and MCP-1 luciferase-expressing promoter construct (MCP-1-LUC), as measured by RLU (relative luminescence units). Performed in triplicate, repeated three times. (I) MCP-1 levels as measured by ELISA in the supernatant of sort purified AdPCs from 1 week HFD-fed mice. n = 3, each group including 6–8 pooled mice pooled. Shown are mean values ± SEM, *p
Figure Legend Snippet: Id3 promotes HFD-induced MCP-1 in VAT . (A, B, G) SVF was isolated from epididymal VAT of 8 to 10 week old Id3 +/+ and Id3 −/− mice fed 1 week of either chow or HFD. n = 7–10. Flow quantitation of MCP-1 hi cells (A) and MCP-1 mid cells (B) per mouse (paired eVAT depots). (C–E) Epididymal VAT and subcutaneous adipose tissue were harvested from 8 to 10 week old Id3 +/+ and Id3 −/− mice fed 4 weeks of either chow or HFD. n = 5–6. (C, D) MCP-1 mRNA levels in epididymal (C) and subcutaneous (D) adipose, represented as fold increase over Id3 +/+ chow. (E) MCP-1 levels as measured by ELISA in the supernatant of epididymal VAT, cultured for 24 h. MCP-1 secretion was normalized per mouse (paired eVAT depots). (F) Weights of epididymal VAT from 8 to 10 week old Id3 +/+ and Id3 −/− mice fed chow-diet or 1 or 4 weeks of HFD. (G) Correlation of quantified MCP-1 hi cells with epididymal VAT weight in 1 week HFD-fed Id3 +/+ and Id3 −/− mice. (H) MCP-1 promoter activity in OP-9 cells, transfected with plasmid encoding ID3 and MCP-1 luciferase-expressing promoter construct (MCP-1-LUC), as measured by RLU (relative luminescence units). Performed in triplicate, repeated three times. (I) MCP-1 levels as measured by ELISA in the supernatant of sort purified AdPCs from 1 week HFD-fed mice. n = 3, each group including 6–8 pooled mice pooled. Shown are mean values ± SEM, *p

Techniques Used: Isolation, Mouse Assay, Flow Cytometry, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Expressing, Construct, Purification

Committed CD45 − CD31 − CD29 + CD34 + Sca-1 + CD24 − AdPCs express and secrete high levels of MCP-1 . Epididymal VAT from 8 to 10 week old C57BL/6J mice was harvested and processed for SVF cells. (A, B) Flow cytometry analysis of CD45 − CD31 − Ter119 − CD29 + CD34 + Sca-1 + AdPCs with representative flow plot (A) and the percentage (B) of AdPCs with MCP-1 hi and MCP-1 mid expression. n = 6 (C) MCP-1 levels as measured by ELISA in the supernatant of equivalent numbers of sort purified AdPCs, total SVF, and Lin + (CD45 + /CD31 + /Ter119 + ) cells. n = 3, each group including 6–8 mice pooled. (D, E) Analysis of MCP-1 hi cells in CD24 + and CD24 − AdPCs with representative plots (D) and quantitation (E) of MCP-1 intracellular staining. n = 15. Shown are mean values ± SEM.
Figure Legend Snippet: Committed CD45 − CD31 − CD29 + CD34 + Sca-1 + CD24 − AdPCs express and secrete high levels of MCP-1 . Epididymal VAT from 8 to 10 week old C57BL/6J mice was harvested and processed for SVF cells. (A, B) Flow cytometry analysis of CD45 − CD31 − Ter119 − CD29 + CD34 + Sca-1 + AdPCs with representative flow plot (A) and the percentage (B) of AdPCs with MCP-1 hi and MCP-1 mid expression. n = 6 (C) MCP-1 levels as measured by ELISA in the supernatant of equivalent numbers of sort purified AdPCs, total SVF, and Lin + (CD45 + /CD31 + /Ter119 + ) cells. n = 3, each group including 6–8 mice pooled. (D, E) Analysis of MCP-1 hi cells in CD24 + and CD24 − AdPCs with representative plots (D) and quantitation (E) of MCP-1 intracellular staining. n = 15. Shown are mean values ± SEM.

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Purification, Quantitation Assay, Staining

Adoptive transfer of Id3 +/+ AdPCs restores HFD-induced MCP-1 expression and M1 macrophage accumulation in Id3 −/− mice (A) Setup of i.p. injection of vehicle or 50,000 sort-purified AdPCs from 2 week HFD-fed Id3 +/+ mice or Id3 −/− mice into Id3 −/− recipient mice. n = 7. After 72 h, recipient mice were fed HFD. GTT was performed after 6 weeks of HFD, and mice were sacrificed after 8 weeks of HFD. (B–C) GTT performed after 6 weeks of HFD. (B) Blood glucose measurements, with asterisks denoting comparison at individual time points. + signifies comparison of vehicle to Id3 +/+ cells and * signifies comparison of Id3 +/+ cells to Id3 −/− cells (C) Area under the curve measurements. (D) Weight gain over 8 weeks of HFD, with asterisks denoting comparison of vehicle to Id3 +/+ at individual time points. (E) Epididymal weights at sacrifice. (F) MCP-1 levels as measured by ELISA in the supernatant of epididymal VAT, cultured for 24 h. MCP-1 secretion was normalized per paired depots. (G–I) Flow quantitation of F4/80 + CD11c + CD206 − M1 macrophages (G) and F4/80 + CD11c − CD206 + M2 macrophages (H) per gram of fat and ratio of M1:M2 macrophages (I). Shown are mean values ± SEM, * or + p
Figure Legend Snippet: Adoptive transfer of Id3 +/+ AdPCs restores HFD-induced MCP-1 expression and M1 macrophage accumulation in Id3 −/− mice (A) Setup of i.p. injection of vehicle or 50,000 sort-purified AdPCs from 2 week HFD-fed Id3 +/+ mice or Id3 −/− mice into Id3 −/− recipient mice. n = 7. After 72 h, recipient mice were fed HFD. GTT was performed after 6 weeks of HFD, and mice were sacrificed after 8 weeks of HFD. (B–C) GTT performed after 6 weeks of HFD. (B) Blood glucose measurements, with asterisks denoting comparison at individual time points. + signifies comparison of vehicle to Id3 +/+ cells and * signifies comparison of Id3 +/+ cells to Id3 −/− cells (C) Area under the curve measurements. (D) Weight gain over 8 weeks of HFD, with asterisks denoting comparison of vehicle to Id3 +/+ at individual time points. (E) Epididymal weights at sacrifice. (F) MCP-1 levels as measured by ELISA in the supernatant of epididymal VAT, cultured for 24 h. MCP-1 secretion was normalized per paired depots. (G–I) Flow quantitation of F4/80 + CD11c + CD206 − M1 macrophages (G) and F4/80 + CD11c − CD206 + M2 macrophages (H) per gram of fat and ratio of M1:M2 macrophages (I). Shown are mean values ± SEM, * or + p

Techniques Used: Adoptive Transfer Assay, Expressing, Mouse Assay, Injection, Purification, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry, Quantitation Assay

Human omental adipocyte progenitor cells express abundant MCP-1: an effect marked by high levels of CD44 . (A–E) Subcutaneous and omental adipose tissue were collected during bariatric surgery from consenting human subjects (n = 14), and were processed to SVF cells for flow cytometry. (A) Percentage of SVF cells that were CD45 − CD31 − CD34 + CD90 + CD44 + adipocyte progenitor cells in human omental and subcutaneous adipose tissue. (B) Percentage of AdPCs that were MCP-1 + in omental and subcutaneous adipose tissue. (C) Representative plot depicting the heterogeneity of CD44 staining in CD45 − CD31 − CD34 + CD90 + cells from omental VAT, and MCP-1 intracellular staining in each CD44 subset. 1 = CD44 − , 2 = CD44 lo , 3 = CD44 hi (D) Percentage of AdPCs that are MCP-1 + as a function of CD44 hi and CD44 lo status in omental and subcutaneous adipose tissue. (E) Correlation of gMFI of MCP-1 with gMFI of CD44 in CD45 − CD31 − CD34 + CD90 + AdPCs in both omental and subcutaneous adipose tissue. Shown are mean values ± SD. (F, G) SVF was isolated from epididymal VAT of 8 to 10 week old C57BL/6J mice fed 1 week of either chow or HFD. n = 4. (F) Percentages of CD45 − CD31 − CD34 + CD29 + Sca-1 + CD24 − AdPCs that are MCP-1 hi as a function of CD44 hi and CD44 lo status, in chow-fed and HFD-fed mice. (G) Correlation of gMFI of MCP-1 with gMFI of CD44 in CD45 − CD31 − CD34 + CD29 + Sca-1 + CD24 − AdPCs, fed chow or HFD. Shown are mean values ± SEM, *p
Figure Legend Snippet: Human omental adipocyte progenitor cells express abundant MCP-1: an effect marked by high levels of CD44 . (A–E) Subcutaneous and omental adipose tissue were collected during bariatric surgery from consenting human subjects (n = 14), and were processed to SVF cells for flow cytometry. (A) Percentage of SVF cells that were CD45 − CD31 − CD34 + CD90 + CD44 + adipocyte progenitor cells in human omental and subcutaneous adipose tissue. (B) Percentage of AdPCs that were MCP-1 + in omental and subcutaneous adipose tissue. (C) Representative plot depicting the heterogeneity of CD44 staining in CD45 − CD31 − CD34 + CD90 + cells from omental VAT, and MCP-1 intracellular staining in each CD44 subset. 1 = CD44 − , 2 = CD44 lo , 3 = CD44 hi (D) Percentage of AdPCs that are MCP-1 + as a function of CD44 hi and CD44 lo status in omental and subcutaneous adipose tissue. (E) Correlation of gMFI of MCP-1 with gMFI of CD44 in CD45 − CD31 − CD34 + CD90 + AdPCs in both omental and subcutaneous adipose tissue. Shown are mean values ± SD. (F, G) SVF was isolated from epididymal VAT of 8 to 10 week old C57BL/6J mice fed 1 week of either chow or HFD. n = 4. (F) Percentages of CD45 − CD31 − CD34 + CD29 + Sca-1 + CD24 − AdPCs that are MCP-1 hi as a function of CD44 hi and CD44 lo status, in chow-fed and HFD-fed mice. (G) Correlation of gMFI of MCP-1 with gMFI of CD44 in CD45 − CD31 − CD34 + CD29 + Sca-1 + CD24 − AdPCs, fed chow or HFD. Shown are mean values ± SEM, *p

Techniques Used: Flow Cytometry, Cytometry, Staining, Isolation, Mouse Assay

1 week of HFD promotes proliferation of AdPCs . (A, B, E) SVF was isolated from epididymal VAT of 8 to 10 week old C57BL/6J mice fed 1 week of either chow or HFD. n = 10–11 per group. (A) Flow quantitation of MCP-1 hi and MCP-1 mid cells per mouse (paired eVAT depots). (B) Flow quantitation of total AdPCs per mouse (paired eVAT depots). (C–D) 7 to 8 week old male C57BL/6J mice were fed standard chow or HFD for 1 week, and were injected with BrdU (bromodeoxyuridine) 5 times over the course of the diet. n = 11. (C) Time course of BrdU injections during 1 week of diet. (D) Percentage of BrdU uptake in AdPCs. (E) Flow quantitation of CD24 + and CD24 − AdPCs per mouse (paired eVAT depots). Shown are mean values ± SEM, **p
Figure Legend Snippet: 1 week of HFD promotes proliferation of AdPCs . (A, B, E) SVF was isolated from epididymal VAT of 8 to 10 week old C57BL/6J mice fed 1 week of either chow or HFD. n = 10–11 per group. (A) Flow quantitation of MCP-1 hi and MCP-1 mid cells per mouse (paired eVAT depots). (B) Flow quantitation of total AdPCs per mouse (paired eVAT depots). (C–D) 7 to 8 week old male C57BL/6J mice were fed standard chow or HFD for 1 week, and were injected with BrdU (bromodeoxyuridine) 5 times over the course of the diet. n = 11. (C) Time course of BrdU injections during 1 week of diet. (D) Percentage of BrdU uptake in AdPCs. (E) Flow quantitation of CD24 + and CD24 − AdPCs per mouse (paired eVAT depots). Shown are mean values ± SEM, **p

Techniques Used: Isolation, Mouse Assay, Flow Cytometry, Quantitation Assay, Injection

CD45 − CD34 + SVF cells in VAT express high levels of MCP-1 . (A–D) SVF and adipocytes were isolated from epididymal VAT of 8 to 10 week old C57BL/6J mice fed 1 week of either chow or HFD, and were analyzed for MCP-1 production. (A–C) MCP-1 mRNA levels in SVF cells and adipocytes, represented as fold increase over chow (A, B) and comparison between populations from HFD-fed mice (C). n = 10. (D) MCP-1 levels in the supernatant from SVF cells and adipocytes of 1 week HFD-fed C57BL/6J mice, cultured for 24 h n = 5. (E, F) Flow cytometry analysis of SVF cells from VAT of 8 to 10 week old C57BL/6J mice, n = 15. (E) Representative flow plot of intracellular MCP-1 staining in SVF. 1 = MCP-1 mid , 2 = MCP-1 hi . (F) Characterization of MCP-1 hi and MCP-1 mid cells using CD45, CD34, F4/80 and CD11b surface staining. 71.6 ± 1.5% of MCP-1 hi cells were CD45 − CD34 + and 66.2 ± 2.1% of MCP-1 mid cells were CD45 + F4/80 + CD11b + . Shown are mean values ± SEM, *p
Figure Legend Snippet: CD45 − CD34 + SVF cells in VAT express high levels of MCP-1 . (A–D) SVF and adipocytes were isolated from epididymal VAT of 8 to 10 week old C57BL/6J mice fed 1 week of either chow or HFD, and were analyzed for MCP-1 production. (A–C) MCP-1 mRNA levels in SVF cells and adipocytes, represented as fold increase over chow (A, B) and comparison between populations from HFD-fed mice (C). n = 10. (D) MCP-1 levels in the supernatant from SVF cells and adipocytes of 1 week HFD-fed C57BL/6J mice, cultured for 24 h n = 5. (E, F) Flow cytometry analysis of SVF cells from VAT of 8 to 10 week old C57BL/6J mice, n = 15. (E) Representative flow plot of intracellular MCP-1 staining in SVF. 1 = MCP-1 mid , 2 = MCP-1 hi . (F) Characterization of MCP-1 hi and MCP-1 mid cells using CD45, CD34, F4/80 and CD11b surface staining. 71.6 ± 1.5% of MCP-1 hi cells were CD45 − CD34 + and 66.2 ± 2.1% of MCP-1 mid cells were CD45 + F4/80 + CD11b + . Shown are mean values ± SEM, *p

Techniques Used: Isolation, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry, Staining

28) Product Images from "Adipose-Derived Stem-Cell-Seeded Non-Cross-Linked Porcine Acellular Dermal Matrix Increases Cellular Infiltration, Vascular Infiltration, and Mechanical Strength of Ventral Hernia Repairs"

Article Title: Adipose-Derived Stem-Cell-Seeded Non-Cross-Linked Porcine Acellular Dermal Matrix Increases Cellular Infiltration, Vascular Infiltration, and Mechanical Strength of Ventral Hernia Repairs

Journal: Tissue Engineering. Part A

doi: 10.1089/ten.tea.2014.0235

Representative analysis of stained cells isolated from cultured rat adipose tissue at P4 (A–E) and P13 (F–J) passages analyzed by fluorescence-assisted cell sorting. P4: (A) 96.0% of cultured adipose-derived stem cells (ASCs) expressed both CD29 and CD90, (B) 97.9% of the cells expressed CD44 and CD90, (C) 9.7% expressed both CD31 and CD90, (D) 1.1% expressed both CD45 and CD90, and (E) 96.1% expressed prolyl 4-hydroxylase, beta (P4HB). P13: (F) 97.5% of cultured ASC expressed both CD29 and CD90, (G) 54.7% of the cells expressed CD44 and CD90, (H) 7.9% expressed both CD31 and CD90, (I) 15.8% expressed both CD45 and CD90, and (J)
Figure Legend Snippet: Representative analysis of stained cells isolated from cultured rat adipose tissue at P4 (A–E) and P13 (F–J) passages analyzed by fluorescence-assisted cell sorting. P4: (A) 96.0% of cultured adipose-derived stem cells (ASCs) expressed both CD29 and CD90, (B) 97.9% of the cells expressed CD44 and CD90, (C) 9.7% expressed both CD31 and CD90, (D) 1.1% expressed both CD45 and CD90, and (E) 96.1% expressed prolyl 4-hydroxylase, beta (P4HB). P13: (F) 97.5% of cultured ASC expressed both CD29 and CD90, (G) 54.7% of the cells expressed CD44 and CD90, (H) 7.9% expressed both CD31 and CD90, (I) 15.8% expressed both CD45 and CD90, and (J)

Techniques Used: Staining, Isolation, Cell Culture, Fluorescence, FACS, Derivative Assay

29) Product Images from "Gaucher disease iPSC-derived osteoblasts have developmental and lysosomal defects that impair bone matrix deposition"

Article Title: Gaucher disease iPSC-derived osteoblasts have developmental and lysosomal defects that impair bone matrix deposition

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddx442

Characterization of mesenchymal stem cells and osteoblasts derived from control and GD iPSC. ( A ) Flow cytometry analysis of iPSC-derived control MSC. Scatter plots show staining for the specific markers of MSC, CD29, CD44 and HLA-ABC, and staining with anti-CD45 as a negative control. Isotype controls are shown at the left. ( B ) qRT-PCR analysis showing the expression of osteoblast markers in iPSC-derived control and GD MSC and osteoblasts as indicated. Results are expressed as fold-change of each osteoblast line compared with its corresponding MSC line (mean ± SEM). P values for control, GD2a and GD3a for each marker are as follows: ALP (0.003, 0.016 and 0.002), Col1 (0.002, 0.372 and 0.049), RUNX2 (0.020, 0.034 and 0.049). ( C ) Alkaline phosphatase stain in control and GD2 osteoblasts. ( D ) Alizarin red stain showing the mineral deposits in control and GD2 osteoblast cultures. Scale bar, 50 µm.
Figure Legend Snippet: Characterization of mesenchymal stem cells and osteoblasts derived from control and GD iPSC. ( A ) Flow cytometry analysis of iPSC-derived control MSC. Scatter plots show staining for the specific markers of MSC, CD29, CD44 and HLA-ABC, and staining with anti-CD45 as a negative control. Isotype controls are shown at the left. ( B ) qRT-PCR analysis showing the expression of osteoblast markers in iPSC-derived control and GD MSC and osteoblasts as indicated. Results are expressed as fold-change of each osteoblast line compared with its corresponding MSC line (mean ± SEM). P values for control, GD2a and GD3a for each marker are as follows: ALP (0.003, 0.016 and 0.002), Col1 (0.002, 0.372 and 0.049), RUNX2 (0.020, 0.034 and 0.049). ( C ) Alkaline phosphatase stain in control and GD2 osteoblasts. ( D ) Alizarin red stain showing the mineral deposits in control and GD2 osteoblast cultures. Scale bar, 50 µm.

Techniques Used: Derivative Assay, Flow Cytometry, Cytometry, Staining, Negative Control, Quantitative RT-PCR, Expressing, Marker, ALP Assay

30) Product Images from "Differential expression of microRNAs in decidua-derived mesenchymal stem cells from patients with pre-eclampsia"

Article Title: Differential expression of microRNAs in decidua-derived mesenchymal stem cells from patients with pre-eclampsia

Journal: Journal of Biomedical Science

doi: 10.1186/s12929-014-0081-3

Identification of dMSCs derived from patients with PE and healthy donors. A : Morphology of dMSCs from healthy pregnant women within 1 week of culture; B : Morphology of dMSCs from patients with PE within 1 week of culture; C : Flow cytometric characterization of dMSCs isolated from healthy pregnant woman during passage 3. Expression of surface antigens CD105, CD73, CD90, CD29, CD44, HLA-DR, CD19, CD11b, CD106, CD45, CD14, CD34, and CD31 was detected using flow cytometry. The percentage of each positive marker is shown. The percentages are shown as mean ± SE from five healthy pregnant women; D : Flow cytometric characterization of dMSCs isolated from patient with PE during passage 3. Expression of surface antigens CD105, CD73, CD90, CD29, CD44, HLA-DR, CD19, CD11b, CD106, CD45, CD14, CD34, and CD31 was detected using flow cytometry. The percentages are shown as mean ± SE from five patients with PE. dMSC, decidua-derived mesenchymal stem cell; PE, pre-eclampsia; SE, standard error.
Figure Legend Snippet: Identification of dMSCs derived from patients with PE and healthy donors. A : Morphology of dMSCs from healthy pregnant women within 1 week of culture; B : Morphology of dMSCs from patients with PE within 1 week of culture; C : Flow cytometric characterization of dMSCs isolated from healthy pregnant woman during passage 3. Expression of surface antigens CD105, CD73, CD90, CD29, CD44, HLA-DR, CD19, CD11b, CD106, CD45, CD14, CD34, and CD31 was detected using flow cytometry. The percentage of each positive marker is shown. The percentages are shown as mean ± SE from five healthy pregnant women; D : Flow cytometric characterization of dMSCs isolated from patient with PE during passage 3. Expression of surface antigens CD105, CD73, CD90, CD29, CD44, HLA-DR, CD19, CD11b, CD106, CD45, CD14, CD34, and CD31 was detected using flow cytometry. The percentages are shown as mean ± SE from five patients with PE. dMSC, decidua-derived mesenchymal stem cell; PE, pre-eclampsia; SE, standard error.

Techniques Used: Derivative Assay, Flow Cytometry, Isolation, Expressing, Cytometry, Marker

31) Product Images from "Regeneration of Articular Cartilage by Human ESC‐Derived Mesenchymal Progenitors Treated Sequentially with BMP‐2 and Wnt5a"

Article Title: Regeneration of Articular Cartilage by Human ESC‐Derived Mesenchymal Progenitors Treated Sequentially with BMP‐2 and Wnt5a

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2016-0020

Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.
Figure Legend Snippet: Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.

Techniques Used: Derivative Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence, Polymerase Chain Reaction, Staining, Cell Culture, ALP Assay, FACS

32) Product Images from "Expression of a Distinct Set of Chemokine Receptors in Adipose Tissue-Derived Stem Cells is Responsible for In Vitro Migration Toward Chemokines Appearing in the Major Pelvic Ganglion Following Cavernous Nerve Injury"

Article Title: Expression of a Distinct Set of Chemokine Receptors in Adipose Tissue-Derived Stem Cells is Responsible for In Vitro Migration Toward Chemokines Appearing in the Major Pelvic Ganglion Following Cavernous Nerve Injury

Journal: Sexual Medicine

doi: 10.1002/sm2.1

(A) Flow cytometry analysis on ADSCs isolated from female human donors shows the vast majority of cells express an CD14−, CD34−, CD45−, HLA-DR− and CD29+, CD90+, CD166+, CD73+, CD105+, CD44+ HLA-ABC+ phenotype, consistent with the typical mesenchymal multipotent stromal cell surface marker profile (light gray area: isotype control, black line: sample). (B) Cultured human ADSCs display a typical fibroblast-like morphology and are able to differentiate toward adipogenic and osteogenic lineages in vitro. Figure 1 is adapted from reference [ 14 ] with permission. ADSC = adipose tissue-derived stem cell.
Figure Legend Snippet: (A) Flow cytometry analysis on ADSCs isolated from female human donors shows the vast majority of cells express an CD14−, CD34−, CD45−, HLA-DR− and CD29+, CD90+, CD166+, CD73+, CD105+, CD44+ HLA-ABC+ phenotype, consistent with the typical mesenchymal multipotent stromal cell surface marker profile (light gray area: isotype control, black line: sample). (B) Cultured human ADSCs display a typical fibroblast-like morphology and are able to differentiate toward adipogenic and osteogenic lineages in vitro. Figure 1 is adapted from reference [ 14 ] with permission. ADSC = adipose tissue-derived stem cell.

Techniques Used: Flow Cytometry, Cytometry, Isolation, Marker, Cell Culture, In Vitro, Derivative Assay

33) Product Images from "A p38 MAPK-Mediated Alteration of COX-2/PGE2 Regulates Immunomodulatory Properties in Human Mesenchymal Stem Cell Aging"

Article Title: A p38 MAPK-Mediated Alteration of COX-2/PGE2 Regulates Immunomodulatory Properties in Human Mesenchymal Stem Cell Aging

Journal: PLoS ONE

doi: 10.1371/journal.pone.0102426

Characterization of hMSCs. (A–C) Images of differentiated hMSCs after induction into specific tissues. (A) The lipid droplet accumulation in differentiated cells was visualized using Oil Red O staining after 2 weeks of adipogenic induction. (B) Calcium deposition was stained with Alizarin Red S after 2 weeks of osteogenic induction. (C) Glycosaminoglycans in cell pellets were revealed by Toluidine blue staining after 2 weeks of chondrogenic induction. (D) hMSCs (1×10 6 cells/ml) were stained with FITC- or PE- conjugated antibodies specific for human CD29, CD34, CD45, CD73, CD105, and HLA-DR.
Figure Legend Snippet: Characterization of hMSCs. (A–C) Images of differentiated hMSCs after induction into specific tissues. (A) The lipid droplet accumulation in differentiated cells was visualized using Oil Red O staining after 2 weeks of adipogenic induction. (B) Calcium deposition was stained with Alizarin Red S after 2 weeks of osteogenic induction. (C) Glycosaminoglycans in cell pellets were revealed by Toluidine blue staining after 2 weeks of chondrogenic induction. (D) hMSCs (1×10 6 cells/ml) were stained with FITC- or PE- conjugated antibodies specific for human CD29, CD34, CD45, CD73, CD105, and HLA-DR.

Techniques Used: Staining

34) Product Images from "Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks"

Article Title: Cryopreservation of human vascular umbilical cord cells under good manufacturing practice conditions for future cell banks

Journal: Journal of Translational Medicine

doi: 10.1186/1479-5876-10-98

Expression of cellular marker molecules by human umbilical vein endothelial cells (HUVEC). Using indirect immunofluorescence staining, highly positive signals (green) were detected for A ) CD31 of fresh cultivated cells and B ) CD31 of cryopreserved cells, E ) von Willebrandt factor (vWF) of fresh cultivated cells and F ) vWF of cryopreserved cells. The presence of C ) CD31 (green) and G ) vWF (green) was detected in the endothelium of native human umbilical cord veins, serving as a control. Immunohistochemical staining verified the presence of D ) CD31 (red) and H ) vWF (red) in the endothelium of native human umbilical cord veins. Using flow cytometry analysis, cellular marker expression of short-term (group A, n = 4) and long-term (group B, n = 4) cryopreserved cells from primary cultures (passage 0) was studied directly after I ) thawing and J ) in passage 3 of recultivation. By comparison, non-cryopreserved fresh cells (n = 3) from I) primary cultures and J) passage 3 were analyzed in parallel as a control group. Using indirect immunofluorescence staining, highly positive signals (green) were also detected for the cellular markers K ) CD144 of fresh cultivated cells and L ) CD144 of cryopreserved cells, M ) endothelial nitric oxide-synthase (eNOS) of fresh cultivated cells and N ) eNOS of cryopreserved cells. Cell nuclei staining is pictured in blue, present in A-H and K-N. All studies of marker expression are exemplarily shown for cells of passage 3.
Figure Legend Snippet: Expression of cellular marker molecules by human umbilical vein endothelial cells (HUVEC). Using indirect immunofluorescence staining, highly positive signals (green) were detected for A ) CD31 of fresh cultivated cells and B ) CD31 of cryopreserved cells, E ) von Willebrandt factor (vWF) of fresh cultivated cells and F ) vWF of cryopreserved cells. The presence of C ) CD31 (green) and G ) vWF (green) was detected in the endothelium of native human umbilical cord veins, serving as a control. Immunohistochemical staining verified the presence of D ) CD31 (red) and H ) vWF (red) in the endothelium of native human umbilical cord veins. Using flow cytometry analysis, cellular marker expression of short-term (group A, n = 4) and long-term (group B, n = 4) cryopreserved cells from primary cultures (passage 0) was studied directly after I ) thawing and J ) in passage 3 of recultivation. By comparison, non-cryopreserved fresh cells (n = 3) from I) primary cultures and J) passage 3 were analyzed in parallel as a control group. Using indirect immunofluorescence staining, highly positive signals (green) were also detected for the cellular markers K ) CD144 of fresh cultivated cells and L ) CD144 of cryopreserved cells, M ) endothelial nitric oxide-synthase (eNOS) of fresh cultivated cells and N ) eNOS of cryopreserved cells. Cell nuclei staining is pictured in blue, present in A-H and K-N. All studies of marker expression are exemplarily shown for cells of passage 3.

Techniques Used: Expressing, Marker, Immunofluorescence, Staining, Immunohistochemistry, Flow Cytometry, Cytometry

35) Product Images from "Regeneration of Articular Cartilage by Human ESC‐Derived Mesenchymal Progenitors Treated Sequentially with BMP‐2 and Wnt5a"

Article Title: Regeneration of Articular Cartilage by Human ESC‐Derived Mesenchymal Progenitors Treated Sequentially with BMP‐2 and Wnt5a

Journal: Stem Cells Translational Medicine

doi: 10.5966/sctm.2016-0020

Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.
Figure Legend Snippet: Mesenchymal stem cell‐like progenitors derived from H9 hESCs (H9‐derived MSCs) share common features with bone marrow‐derived MSCs. (A): Expression of cell surface antigens on progenitor cells by flow cytometry analysis. Histograms display percent cells expressing cell‐surface antigens of mesenchymal markers on H9‐derived MSC‐like cells (H9‐MSCs). H9‐MSCs express markers associated with the mesenchymal phenotype (CD44, CD73, CD29, CD166, CD90, CD105, and HLA‐ABC) and lack expression of endothelial (CD31) and hematopoietic markers (CD45 and HLA‐DR). Negative isotype controls are shown with percent fluorescence indicated for positive signal over isotype controls. (B): Comparative flow cytometry analyses of H9‐derived MSCs and the adult human bone marrow‐derived MSCs (Lonza) showed similar cell surface expression profiles. (C): Quantitative reverse‐transcriptase polymerase chain reaction analyses revealed downregulation of canonical pluripotency genes in H9‐derived MSC progenitors compared with undifferentiated H9 ESCs. (D): Alcian Blue staining of H9‐derived MSC high‐density pellets cultured in serum‐free chondrogenic medium for 21 days (magnification, ×4); Oil Red O staining of H9‐derived MSC progenitor monolayer cells cultured in adipogenic medium for 21 days (magnification, ×20); and a 10‐cm tissue culture dish (magnification, ×1) with alkaline phosphatase staining of H9‐derived MSC monolayer cells cultured in osteogenic medium for 21 days. Abbreviations: ALP, alkaline phosphatase; BM‐MSC, bone marrow‐derived mesenchymal stem cell; ESC, embryonic stem cell; FACS, fluorescence‐activated cell sorting; FITC‐A, fluorescein isothiocyanate A; hESC, human embryonic stem cell; HLA‐ABC, human leukocyte antigen ABC; Max, maximum; MSC, mesenchymal stem cell; PE‐A, phycoerythrin A.

Techniques Used: Derivative Assay, Expressing, Flow Cytometry, Cytometry, Fluorescence, Polymerase Chain Reaction, Staining, Cell Culture, ALP Assay, FACS

36) Product Images from "A Therapeutic Strategy for Spinal Cord Defect: Human Dental Follicle Cells Combined with Aligned PCL/PLGA Electrospun Material"

Article Title: A Therapeutic Strategy for Spinal Cord Defect: Human Dental Follicle Cells Combined with Aligned PCL/PLGA Electrospun Material

Journal: BioMed Research International

doi: 10.1155/2015/197183

Flow cytometric analyses for immunophenotypic characteristics of hDFCs. hDFCs were positive for CD29, CD44, CD90, CD105, CD146, and CD166 and negative for CD24, CD34, and CD45. Green color stands for being labeled by FITC and red color stands for being labeled by PE.
Figure Legend Snippet: Flow cytometric analyses for immunophenotypic characteristics of hDFCs. hDFCs were positive for CD29, CD44, CD90, CD105, CD146, and CD166 and negative for CD24, CD34, and CD45. Green color stands for being labeled by FITC and red color stands for being labeled by PE.

Techniques Used: Flow Cytometry, Labeling

37) Product Images from "Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation"

Article Title: Prospectively isolated mesenchymal stem/stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation

Journal: Scientific Reports

doi: 10.1038/s41598-017-05099-1

Differentiation ability and lack of infiltration of CD73 + rMSCs. ( a ) Capacity of CD73-MSCs to differentiate into adipocytes, chondrocytes, and osteoblasts. Scale bar, 100 µm. ( b ) Double fluorescence staining to label the chondrogenic marker aggrecan (green) and the macrophage marker Iba1 (red) (top). Scale bar, 100 µm. Histological sections of cell pellets transplanted after 2 weeks. The pellets stained with safranin O (low). Scale bar, 100 µm. ( c ) Quantitative analysis of the mRNA expression of Cxcl12 , Ccl2 , Il10 , and Tgf b1 in freshly isolated MSCs (CD73 + and CD73 − cells). The mRNA expression of each gene was normalized to that of Hprt. The gene expression in CD73 − CD45 − CD31 − cells was set as 1.0.
Figure Legend Snippet: Differentiation ability and lack of infiltration of CD73 + rMSCs. ( a ) Capacity of CD73-MSCs to differentiate into adipocytes, chondrocytes, and osteoblasts. Scale bar, 100 µm. ( b ) Double fluorescence staining to label the chondrogenic marker aggrecan (green) and the macrophage marker Iba1 (red) (top). Scale bar, 100 µm. Histological sections of cell pellets transplanted after 2 weeks. The pellets stained with safranin O (low). Scale bar, 100 µm. ( c ) Quantitative analysis of the mRNA expression of Cxcl12 , Ccl2 , Il10 , and Tgf b1 in freshly isolated MSCs (CD73 + and CD73 − cells). The mRNA expression of each gene was normalized to that of Hprt. The gene expression in CD73 − CD45 − CD31 − cells was set as 1.0.

Techniques Used: Fluorescence, Staining, Marker, Expressing, Isolation

Morphological and self-renewal evaluation by clonal analysis. ( a ) Representative flow cytometric profiles of rBM cells stained for CD29 and CD54. PI − , CD45 − , and CD31 − cells were gated. Baseline CD29 and CD54 expression is shown in pink. Sorted subsets are shown in black dotted squares. The cell number ratio is shown in the profile. ( b ) Morphological appearance of sorted sub-population cells. Scale bar, 100 µm. ( c ) Single-cell sorting assay of rBM cell sub-populations. WBM cells and four cell populations (CD29 + /CD54 + , CD29 + /CD54 − , CD29 − /CD54 + , and CD29 − /CD54 − ) were sorted into 96-well plates, and the number of colonies formed was counted on day 10. Scale bar, 500 µm.
Figure Legend Snippet: Morphological and self-renewal evaluation by clonal analysis. ( a ) Representative flow cytometric profiles of rBM cells stained for CD29 and CD54. PI − , CD45 − , and CD31 − cells were gated. Baseline CD29 and CD54 expression is shown in pink. Sorted subsets are shown in black dotted squares. The cell number ratio is shown in the profile. ( b ) Morphological appearance of sorted sub-population cells. Scale bar, 100 µm. ( c ) Single-cell sorting assay of rBM cell sub-populations. WBM cells and four cell populations (CD29 + /CD54 + , CD29 + /CD54 − , CD29 − /CD54 + , and CD29 − /CD54 − ) were sorted into 96-well plates, and the number of colonies formed was counted on day 10. Scale bar, 500 µm.

Techniques Used: Flow Cytometry, Staining, Expressing, FACS

38) Product Images from "Prevascularization of collagen-glycosaminoglycan scaffolds: stromal vascular fraction versus adipose tissue-derived microvascular fragments"

Article Title: Prevascularization of collagen-glycosaminoglycan scaffolds: stromal vascular fraction versus adipose tissue-derived microvascular fragments

Journal: Journal of Biological Engineering

doi: 10.1186/s13036-018-0118-3

Incorporation and vascularization of implanted scaffolds. a-d HE-stained sections of SVF- ( a , b ) and ad-MVF-seeded ( c , d ) Integra® scaffolds on day 14 after implantation into full-thickness skin defects within dorsal skinfold chambers of C57BL/6 recipient mice (broken lines = implant; closed frames = center zones of the implants; b , d = higher magnifications of closed frames in a and c arrows = nuclei of individual cells). Scale bars: a , c = 260 μm; b , d = 40 μm. e-g Polarized light microscopy of Sirius red-stained sections of normal skin ( e ) as well as SVF- ( f ) and ad-MVF-seeded ( g ) Integra® scaffolds. Scale bars: 25 μm. h Total collagen ratio in the border and center zones of SVF- (white bars, n = 8) and ad-MVF-seeded (black bars, n = 8) Integra® scaffolds on day 14 after implantation, as assessed by histology. Means ± SEM. i-l Immunohistochemical detection of CD31 + microvessels (arrows) within the border ( i , j ) and center ( k , l ) zones of SVF- ( i , k ) and ad-MVF-seeded ( j, l ) Integra® scaffolds. Scale bars: 25 μm. m Microvessel density in the border and center zones of SVF- (white bars, n = 8) and ad-MVF-seeded (black bars, n = 8) Integra® scaffolds on day 14 after implantation, as assessed by immunohistochemistry. Means ± SEM. * p
Figure Legend Snippet: Incorporation and vascularization of implanted scaffolds. a-d HE-stained sections of SVF- ( a , b ) and ad-MVF-seeded ( c , d ) Integra® scaffolds on day 14 after implantation into full-thickness skin defects within dorsal skinfold chambers of C57BL/6 recipient mice (broken lines = implant; closed frames = center zones of the implants; b , d = higher magnifications of closed frames in a and c arrows = nuclei of individual cells). Scale bars: a , c = 260 μm; b , d = 40 μm. e-g Polarized light microscopy of Sirius red-stained sections of normal skin ( e ) as well as SVF- ( f ) and ad-MVF-seeded ( g ) Integra® scaffolds. Scale bars: 25 μm. h Total collagen ratio in the border and center zones of SVF- (white bars, n = 8) and ad-MVF-seeded (black bars, n = 8) Integra® scaffolds on day 14 after implantation, as assessed by histology. Means ± SEM. i-l Immunohistochemical detection of CD31 + microvessels (arrows) within the border ( i , j ) and center ( k , l ) zones of SVF- ( i , k ) and ad-MVF-seeded ( j, l ) Integra® scaffolds. Scale bars: 25 μm. m Microvessel density in the border and center zones of SVF- (white bars, n = 8) and ad-MVF-seeded (black bars, n = 8) Integra® scaffolds on day 14 after implantation, as assessed by immunohistochemistry. Means ± SEM. * p

Techniques Used: Staining, Mouse Assay, Light Microscopy, Immunohistochemistry

39) Product Images from "Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity"

Article Title: Aberrant expression of Twist1 in diseased articular cartilage and a potential role in the modulation of osteoarthritis severity

Journal: Genes & Diseases

doi: 10.1016/j.gendis.2015.12.005

Expression of Twist1 is repressed in differentiating human articular chondrocytes. (A) De-differentiated normal human articular chondrocytes (monolayer passage 5) exhibited features of mesenchymal-like progenitor cells. Cell surface flow cytometric analyses of the proportion of dedifferentiated human articular chondrocytes expressing CD29, CD44, CD73, CD90, CD105, CD166, HLA-ABC, HLA-DR, CD31, and CD45 receptors. IgG1 antibodies were included as a negative control. (B) Alcian blue staining of human articular chondrocyte pellets cultured in serum-free media and in the presence of Bmp-2 for 21 days. Higher magnification images in (ii) and (iii) revealed a heterogeneous population of immature and hypertrophic chondrocytes, respectively. Scale bar, 100 um. (C) RT-PCR-based gene expression analyses of Twist1 and chondrogenic markers Sox9, Runx2, Col2a1, Col9a1 and ColXa1 in normal human articular chondrocyte pellets treated with Bmp-2 for 5, 8, 14, and 21 days in culture. The relative mRNA level in each sample is normalized to its Gapdh content. The expression of Twist1 was suppressed as cells acquired a more differentiated chondrocyte phenotype. * p
Figure Legend Snippet: Expression of Twist1 is repressed in differentiating human articular chondrocytes. (A) De-differentiated normal human articular chondrocytes (monolayer passage 5) exhibited features of mesenchymal-like progenitor cells. Cell surface flow cytometric analyses of the proportion of dedifferentiated human articular chondrocytes expressing CD29, CD44, CD73, CD90, CD105, CD166, HLA-ABC, HLA-DR, CD31, and CD45 receptors. IgG1 antibodies were included as a negative control. (B) Alcian blue staining of human articular chondrocyte pellets cultured in serum-free media and in the presence of Bmp-2 for 21 days. Higher magnification images in (ii) and (iii) revealed a heterogeneous population of immature and hypertrophic chondrocytes, respectively. Scale bar, 100 um. (C) RT-PCR-based gene expression analyses of Twist1 and chondrogenic markers Sox9, Runx2, Col2a1, Col9a1 and ColXa1 in normal human articular chondrocyte pellets treated with Bmp-2 for 5, 8, 14, and 21 days in culture. The relative mRNA level in each sample is normalized to its Gapdh content. The expression of Twist1 was suppressed as cells acquired a more differentiated chondrocyte phenotype. * p

Techniques Used: Expressing, Flow Cytometry, Negative Control, Staining, Cell Culture, Reverse Transcription Polymerase Chain Reaction

40) Product Images from "β1-Integrin– and KV1.3 channel–dependent signaling stimulates glutamate release from Th17 cells"

Article Title: β1-Integrin– and KV1.3 channel–dependent signaling stimulates glutamate release from Th17 cells

Journal: The Journal of Clinical Investigation

doi: 10.1172/JCI126381

Intrathecal administration of MgTX ameliorates EAE disease course, and the differentiation of CNS-infiltrating 2D2 cells remains unchanged. ( A ) In mice with MOG 35–55 -induced C57Bl6 EAE, intrathecal injection of MgTX (1.3 ng MgTX/mouse; n = 11 for 2 independent EAE experiments) into the CSF every other day for 14 days, starting on day 7, led to a significant and reproducible reduction in the clinical EAE score compared with the PBS-treated control group ( n = 13). ( B ) In mice with MOG 35–55 -induced C57Bl6 EAE, intrathecal injection of MgTX (1.3 ng MgTX/mouse, n = 8) into the CSF every other day for 14 days, starting after disease onset (treatment started when the EAE score was > 1, on approximately day 12), led to a significant reduction in the clinical EAE score compared with the PBS-treated control group ( n = 8 for 2 independent EAE experiments). ( C ) Intracellular cytokine staining was performed on T lymphocytes ex vivo on day 28. Staining for IL-17 and IFN-γ was analyzed by flow cytometry and is shown as the percentage of CD4 + cells within the CNS and as the MFI of CD4 + cells. n = 4 PBS-treated mice; n = 5 MgTX-treated mice. ( D ) Cryosections of EAE mouse brains were stained for SMI312 ( n = 4–5 sections at least 100 μm apart, from 4 WT and MgTx-treated mice on day 28). Images were acquired using an Olympus microscope equipped with a cellSense camera and analyzed with ImageJ software. Scale bars: 20 μm and 5 μm (enlarged insets). Data indicate the mean ± SEM. * P
Figure Legend Snippet: Intrathecal administration of MgTX ameliorates EAE disease course, and the differentiation of CNS-infiltrating 2D2 cells remains unchanged. ( A ) In mice with MOG 35–55 -induced C57Bl6 EAE, intrathecal injection of MgTX (1.3 ng MgTX/mouse; n = 11 for 2 independent EAE experiments) into the CSF every other day for 14 days, starting on day 7, led to a significant and reproducible reduction in the clinical EAE score compared with the PBS-treated control group ( n = 13). ( B ) In mice with MOG 35–55 -induced C57Bl6 EAE, intrathecal injection of MgTX (1.3 ng MgTX/mouse, n = 8) into the CSF every other day for 14 days, starting after disease onset (treatment started when the EAE score was > 1, on approximately day 12), led to a significant reduction in the clinical EAE score compared with the PBS-treated control group ( n = 8 for 2 independent EAE experiments). ( C ) Intracellular cytokine staining was performed on T lymphocytes ex vivo on day 28. Staining for IL-17 and IFN-γ was analyzed by flow cytometry and is shown as the percentage of CD4 + cells within the CNS and as the MFI of CD4 + cells. n = 4 PBS-treated mice; n = 5 MgTX-treated mice. ( D ) Cryosections of EAE mouse brains were stained for SMI312 ( n = 4–5 sections at least 100 μm apart, from 4 WT and MgTx-treated mice on day 28). Images were acquired using an Olympus microscope equipped with a cellSense camera and analyzed with ImageJ software. Scale bars: 20 μm and 5 μm (enlarged insets). Data indicate the mean ± SEM. * P

Techniques Used: Mouse Assay, Injection, Staining, Ex Vivo, Flow Cytometry, Microscopy, Software

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Flow Cytometry:

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Isolation:

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Cytometry:

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Construct:

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Incubation:

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Expressing:

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Polymerase Chain Reaction:

Article Title: Molecular Profiling of Single Sca-1+/CD34+,− Cells--The Putative Murine Lung Stem Cells
Article Snippet: .. For initial molecular characterization of isolated cells, PCR on transcripts of Sca-1 , CD34 , CD45 and CD31 were performed. .. In order to differentiate between a more epithelial or mesenchymal phenotype of isolated cells, we conducted further PCRs specific for epithelial markers Epcam (Epithelial cell adhesion molecule), Itga (Integrin alpha-6) and Sftpc (Surfactant protein C) and mesenchymal markers CD90 (Thy-1) and Pdgfrα (platelet derived growth factor receptor alpha, CD140a), as suggested by McQualter et al. .

Crocin Bleaching Assay:

Article Title: Multidrug-Resistant Pseudomonas aeruginosa Accelerate Intestinal, Extra-Intestinal, and Systemic Inflammatory Responses in Human Microbiota-Associated Mice With Subacute Ileitis
Article Snippet: .. After 18 h at 37°C, IFN-γ, TNF, MCP-1, IL-6, and IL-10 concentrations were measured in culture supernatants and serum by the Mouse Inflammation Cytometric Bead Assay (CBA; BD Bioscience) applying a BD FACSCanto II flow cytometer (BD Bioscience). ..

Software:

Article Title: Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis
Article Snippet: .. PI (0.1 mg ml−1 ) was then added and samples were allowed to stand 15 min in the dark at room temperature before flow cytometry analysis using CELLQuest software (Becton Dickinson, San Jose, CA, U.S.A.). .. After incubation with actinomycin D, adherent cells were fixed for 10 min with PBS containing 3% paraformaldehyde on ice.

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  • 86
    Becton Dickinson pe mouse anti human cd29
    Combination of Kindlin-2 knockdown and docetaxel treatment has a dramatic inhibitory effect on integrin β1-mediated cell spreading (A, B C) Representative histograms using flow cytometry of PC3 cells with the indicated transfections and staining with PE-conjugated anti <t>CD29</t> antibody for cell surface β1 integrin (A B) and with FITC-conjugated anti CD61 antibody for β3 integrin (C). (D) Western blots with anti-K2 antibody of cell lysates from PC3 cells with the indicated transfections. β-Actin was used as a loading control. (E) Representative micrographs of Alexa 568 phalloidin-stained PC3 cells that were transfected with either a non-targeting siRNA (NT) or Kindlin-2 siRNA and seeded on uncoated coverslips (No Ligand) or on coverslips coated with fibronectin (20μg/ml) for 2 hrs in the presence or absence of docetaxel (Doc). (F) Quantification of PC3 cell spreading under the indicated treatments. Data are the fold-change in cell spreading normalized to the values for untreated and non-targeting siRNA transfected cells. Data are representative of 3 independent experiments (*, p
    Pe Mouse Anti Human Cd29, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 86/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pe mouse anti human cd29/product/Becton Dickinson
    Average 86 stars, based on 3 article reviews
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    pe mouse anti human cd29 - by Bioz Stars, 2020-09
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    85
    Becton Dickinson anti vla 4 antibodies
    Human BM infiltration and treatment-free survival analysis of CLL patients with discordant <t>VLA-4</t> and CD38 risk. (A) Human bone marrow biopsy histology was used to determine the BM lymphoid infiltrate rate of the individual VLA-4/CD38 discordant patients (n = 15). “High”: lymphoid infiltration of human BM greater than 50%, “Low”: ≤ 50%. (B) Kaplan-Meier analysis of treatment-free survival of CLL patients (n = 144) separated by their VLA-4 and CD38 risk (VLA-4-/CD38-, orange line, n = 83; VLA-4+/CD38+, green line, n = 30; VLA-4-/CD38+, blue line, n = 13; VLA-4+/CD38-, red line, n = 18; Logrank test for trend compared to VLA-4-CD38- control-group, p = .0004). When comparing the individual survival curves of all patient groups, all three groups with individual or combined high-risk parameters show significantly shorter treatment free-survival than patient group with VLA-4-/CD38- (VLA-4+/CD38+, p = .0042; VLA-4-/CD38+, p
    Anti Vla 4 Antibodies, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti vla 4 antibodies - by Bioz Stars, 2020-09
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    Combination of Kindlin-2 knockdown and docetaxel treatment has a dramatic inhibitory effect on integrin β1-mediated cell spreading (A, B C) Representative histograms using flow cytometry of PC3 cells with the indicated transfections and staining with PE-conjugated anti CD29 antibody for cell surface β1 integrin (A B) and with FITC-conjugated anti CD61 antibody for β3 integrin (C). (D) Western blots with anti-K2 antibody of cell lysates from PC3 cells with the indicated transfections. β-Actin was used as a loading control. (E) Representative micrographs of Alexa 568 phalloidin-stained PC3 cells that were transfected with either a non-targeting siRNA (NT) or Kindlin-2 siRNA and seeded on uncoated coverslips (No Ligand) or on coverslips coated with fibronectin (20μg/ml) for 2 hrs in the presence or absence of docetaxel (Doc). (F) Quantification of PC3 cell spreading under the indicated treatments. Data are the fold-change in cell spreading normalized to the values for untreated and non-targeting siRNA transfected cells. Data are representative of 3 independent experiments (*, p

    Journal: Molecular cancer research : MCR

    Article Title: miR-138-mediated Regulation of Kindlin-2 Expression Modulates Sensitivity to Chemotherapeutics

    doi: 10.1158/1541-7786.MCR-15-0299

    Figure Lengend Snippet: Combination of Kindlin-2 knockdown and docetaxel treatment has a dramatic inhibitory effect on integrin β1-mediated cell spreading (A, B C) Representative histograms using flow cytometry of PC3 cells with the indicated transfections and staining with PE-conjugated anti CD29 antibody for cell surface β1 integrin (A B) and with FITC-conjugated anti CD61 antibody for β3 integrin (C). (D) Western blots with anti-K2 antibody of cell lysates from PC3 cells with the indicated transfections. β-Actin was used as a loading control. (E) Representative micrographs of Alexa 568 phalloidin-stained PC3 cells that were transfected with either a non-targeting siRNA (NT) or Kindlin-2 siRNA and seeded on uncoated coverslips (No Ligand) or on coverslips coated with fibronectin (20μg/ml) for 2 hrs in the presence or absence of docetaxel (Doc). (F) Quantification of PC3 cell spreading under the indicated treatments. Data are the fold-change in cell spreading normalized to the values for untreated and non-targeting siRNA transfected cells. Data are representative of 3 independent experiments (*, p

    Article Snippet: For the quantification of β1 and β3 integrin surface expression, we used PE mouse anti-human CD29 and FITC mouse anti-human CD61, and their respective PE and FITC mouse IgG controls (BD Pharmigen; Franklin Lakes), NJ.

    Techniques: Flow Cytometry, Cytometry, Transfection, Staining, Western Blot

    Suppression of MSC-MV incorporation into HUVECs by exogenous addition of Anx-V, anti-CD29 and anti-CD44 antibodies. (A) Flow cytometry was used to assess the CFSE intensity after the cells were cultured for 12 hours. CTR: HUVECs without MSC-MVs; MSC-MV: HUVECs with MSC-MVs only. X-axis: the forward scatter corner signals showing the cellular size. Y-axis: CFSE fluorescence intensity; (B) The percentages of the CFSE-positive cells are indicated. *P: vs .CTR

    Journal: PLoS ONE

    Article Title: Surface Phosphatidylserine Is Responsible for the Internalization on Microvesicles Derived from Hypoxia-Induced Human Bone Marrow Mesenchymal Stem Cells into Human Endothelial Cells

    doi: 10.1371/journal.pone.0147360

    Figure Lengend Snippet: Suppression of MSC-MV incorporation into HUVECs by exogenous addition of Anx-V, anti-CD29 and anti-CD44 antibodies. (A) Flow cytometry was used to assess the CFSE intensity after the cells were cultured for 12 hours. CTR: HUVECs without MSC-MVs; MSC-MV: HUVECs with MSC-MVs only. X-axis: the forward scatter corner signals showing the cellular size. Y-axis: CFSE fluorescence intensity; (B) The percentages of the CFSE-positive cells are indicated. *P: vs .CTR

    Article Snippet: MV internalization assessed by flow cytometry HUVECs were cultured in the presence of CFSE-labeled MSC-MVs that had been pretreated with mouse-originated monoclonal antibodies against human CD29 and CD44 (BD, USA) at a dose of 1μg/ml and/or recombinant human Anx-V (Biovision, USA) at graded concentrations.

    Techniques: Flow Cytometry, Cytometry, Cell Culture, Fluorescence

    Human BM infiltration and treatment-free survival analysis of CLL patients with discordant VLA-4 and CD38 risk. (A) Human bone marrow biopsy histology was used to determine the BM lymphoid infiltrate rate of the individual VLA-4/CD38 discordant patients (n = 15). “High”: lymphoid infiltration of human BM greater than 50%, “Low”: ≤ 50%. (B) Kaplan-Meier analysis of treatment-free survival of CLL patients (n = 144) separated by their VLA-4 and CD38 risk (VLA-4-/CD38-, orange line, n = 83; VLA-4+/CD38+, green line, n = 30; VLA-4-/CD38+, blue line, n = 13; VLA-4+/CD38-, red line, n = 18; Logrank test for trend compared to VLA-4-CD38- control-group, p = .0004). When comparing the individual survival curves of all patient groups, all three groups with individual or combined high-risk parameters show significantly shorter treatment free-survival than patient group with VLA-4-/CD38- (VLA-4+/CD38+, p = .0042; VLA-4-/CD38+, p

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: Human BM infiltration and treatment-free survival analysis of CLL patients with discordant VLA-4 and CD38 risk. (A) Human bone marrow biopsy histology was used to determine the BM lymphoid infiltrate rate of the individual VLA-4/CD38 discordant patients (n = 15). “High”: lymphoid infiltration of human BM greater than 50%, “Low”: ≤ 50%. (B) Kaplan-Meier analysis of treatment-free survival of CLL patients (n = 144) separated by their VLA-4 and CD38 risk (VLA-4-/CD38-, orange line, n = 83; VLA-4+/CD38+, green line, n = 30; VLA-4-/CD38+, blue line, n = 13; VLA-4+/CD38-, red line, n = 18; Logrank test for trend compared to VLA-4-CD38- control-group, p = .0004). When comparing the individual survival curves of all patient groups, all three groups with individual or combined high-risk parameters show significantly shorter treatment free-survival than patient group with VLA-4-/CD38- (VLA-4+/CD38+, p = .0042; VLA-4-/CD38+, p

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques:

    VLA-4 and CD38 are higher expressed in BM than in PB. Paired analysis of VLA-4 (A) and CD38 expression (B) on CD19+CD5+ CLL cells in PB and BM samples of patients by flow cytometry (n = 16, Wilcoxon signed rank test, VLA-4: p = .0069, CD38: p = .0362). Cases marked with red boxes in the left panels are drawn on a smaller scale in the right panels. (C) Immunohistochemical analysis of VLA-4 and CD38 expression in BM sections of a low-risk (VLA-4-/CD38-) CLL patient (top), and of a high-risk (VLA-4+/CD38+) patient (bottom) shown with indicated magnitudes. *, P

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: VLA-4 and CD38 are higher expressed in BM than in PB. Paired analysis of VLA-4 (A) and CD38 expression (B) on CD19+CD5+ CLL cells in PB and BM samples of patients by flow cytometry (n = 16, Wilcoxon signed rank test, VLA-4: p = .0069, CD38: p = .0362). Cases marked with red boxes in the left panels are drawn on a smaller scale in the right panels. (C) Immunohistochemical analysis of VLA-4 and CD38 expression in BM sections of a low-risk (VLA-4-/CD38-) CLL patient (top), and of a high-risk (VLA-4+/CD38+) patient (bottom) shown with indicated magnitudes. *, P

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques: Expressing, Flow Cytometry, Cytometry, Immunohistochemistry

    VLA-4 and CD38 expression are associated in CLL cells. Percent VLA-4+ correlated to percent CD38+ CLL cells of 144 CLL patients (Spearman's Rho = .6293, P

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: VLA-4 and CD38 expression are associated in CLL cells. Percent VLA-4+ correlated to percent CD38+ CLL cells of 144 CLL patients (Spearman's Rho = .6293, P

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques: Expressing

    VLA-4, and not CD38, is essential for BM homing. Human CLL cells from different patients were injected into NOD/SCID mice. After 180 min mice were sacrificed and the number of human cells detected by flow cytometry. (A) BM and spleen homing rates of CLL cells from patients separated into four groups according to their VLA-4 and CD38 risk status (VLA-4-/CD38-, n = 9; VLA-4+/CD38+, n = 10; VLA-4-/CD38+, n = 7; VLA-4+/CD38-, n = 10; Anova, Kruskal-Wallis test, BM: p

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: VLA-4, and not CD38, is essential for BM homing. Human CLL cells from different patients were injected into NOD/SCID mice. After 180 min mice were sacrificed and the number of human cells detected by flow cytometry. (A) BM and spleen homing rates of CLL cells from patients separated into four groups according to their VLA-4 and CD38 risk status (VLA-4-/CD38-, n = 9; VLA-4+/CD38+, n = 10; VLA-4-/CD38+, n = 7; VLA-4+/CD38-, n = 10; Anova, Kruskal-Wallis test, BM: p

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques: Injection, Mouse Assay, Flow Cytometry, Cytometry

    CLL cells from patients with different risk-groups are equally protected by stromal cells but cells from high-risk patients are able to adhere in higher numbers. (A) VCAM-1 expression on M2-10B4 cells was detected by flow cytometry. (B) PBMCs from CLL patients were cultured in the absence or presence of M2-10B4 cells for 48 hours. VLA-4 and CD38 expression levels on viable CD19+CD5+ CLL cells were determined by flow cytometry at indicated time points. (n = 5, Repeated Measures ANOVA, VLA-4, p = .0083; CD38, p = .1681; Bonferroni's Multiple Comparision Test) (C-F) PBMCs from CLL patients were co-cultured with M2-10B4 cells and after 48 hours viability and adhesion of CLL cells was determined by flow cytometry. (C) Viability of CLL cells from patients with low (n = 7) and high (n = 7) VLA-4-risk left untreated (Mann Whitney test, p = .9015) or treated with 5 µM fludarabine (Unpaired t-test, p = .4626). (D) Adhesion of CLL cells to M2-10B4 of different patient risk-groups (a, VLA-4, low, n = 7; high, n = 7; Unpaired t-test, p = .0003; b, CD38, low, n = 9; high, n = 5, Unpaired t-test, p = .0560). (E) Adhesion of CLL cells to M2-10B4 cells of VLA-4 low-risk (n = 7) or high-risk (n = 7) patient samples pretreated with isotype control (Control) or anti-VLA-4 antibodies. (Paired t-test, low-risk: p = ,4876; high-risk: p = .0059) (F) Viability of non-adherent and adherent CLL cells to M2-10B4 cells of VLA-4 low-risk (n = 7, Paired t test, p = .1186) or high-risk (n = 7, Wilcoxon signed rank test, p = .5781) patient samples. **, P

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: CLL cells from patients with different risk-groups are equally protected by stromal cells but cells from high-risk patients are able to adhere in higher numbers. (A) VCAM-1 expression on M2-10B4 cells was detected by flow cytometry. (B) PBMCs from CLL patients were cultured in the absence or presence of M2-10B4 cells for 48 hours. VLA-4 and CD38 expression levels on viable CD19+CD5+ CLL cells were determined by flow cytometry at indicated time points. (n = 5, Repeated Measures ANOVA, VLA-4, p = .0083; CD38, p = .1681; Bonferroni's Multiple Comparision Test) (C-F) PBMCs from CLL patients were co-cultured with M2-10B4 cells and after 48 hours viability and adhesion of CLL cells was determined by flow cytometry. (C) Viability of CLL cells from patients with low (n = 7) and high (n = 7) VLA-4-risk left untreated (Mann Whitney test, p = .9015) or treated with 5 µM fludarabine (Unpaired t-test, p = .4626). (D) Adhesion of CLL cells to M2-10B4 of different patient risk-groups (a, VLA-4, low, n = 7; high, n = 7; Unpaired t-test, p = .0003; b, CD38, low, n = 9; high, n = 5, Unpaired t-test, p = .0560). (E) Adhesion of CLL cells to M2-10B4 cells of VLA-4 low-risk (n = 7) or high-risk (n = 7) patient samples pretreated with isotype control (Control) or anti-VLA-4 antibodies. (Paired t-test, low-risk: p = ,4876; high-risk: p = .0059) (F) Viability of non-adherent and adherent CLL cells to M2-10B4 cells of VLA-4 low-risk (n = 7, Paired t test, p = .1186) or high-risk (n = 7, Wilcoxon signed rank test, p = .5781) patient samples. **, P

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques: Expressing, Flow Cytometry, Cytometry, Cell Culture, MANN-WHITNEY

    Ki-67 expression in BM aspirates is higher in CLL samples from high-risk patients, and VLA-4 negative and CD38 positive subclones express higher levels of Ki-67 in BM. MNCs from BM aspirates of CLL patients were stained with anti-CD19-PC7, CD5-PC5 and either VLA-4-PE or CD38-PE or respective isotype control-PE antibody and intracellulary stained with Ki-67-FITC or isotype control before flow cytometric analysis. (A) Left. Difference in Ki-67 expression in CLL samples from VLA-4 low (n = 6) and high-risk (n = 6, Unpaired t-test, p = .0298) patients as well as CD38 low (n = 5) and high-risk patients (n = 7, Unpaired t-test, p = .0278). Right. Difference in Ki-67 expression in CLL cells from patients with a combined low risk (-/-, VLA-4-/CD38-, n = 4), with discordant VLA-4 and CD38 risk (+/− −/+, n = 3), and VLA-4+/CD38+ (+/+, n = 5, ANOVA, One-way analysis of variance, p = .0233, Bonferroni's multiple comparision test). (B) Representative plot of CLL cells (CD19+/CD5+) analysed for their VLA-4 expression. VLA-4 negative and VLA-4 positive CLL cells were gated and further analyzed for their Ki-67 expression. (C) Representative plots for CD38 analysis. Percentage of Ki-67+ CLL cells in (A, right) VLA-4 negative and VLA-4 positive subclones within individual patients (n = 12, Paired t-test, p = .0275) or in (B, right) CD38 negative and CD38 positive subclones (n = 12, Paired t-test, p = .0048). neg, negative subclones; pos, positive subclones. *, P

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: Ki-67 expression in BM aspirates is higher in CLL samples from high-risk patients, and VLA-4 negative and CD38 positive subclones express higher levels of Ki-67 in BM. MNCs from BM aspirates of CLL patients were stained with anti-CD19-PC7, CD5-PC5 and either VLA-4-PE or CD38-PE or respective isotype control-PE antibody and intracellulary stained with Ki-67-FITC or isotype control before flow cytometric analysis. (A) Left. Difference in Ki-67 expression in CLL samples from VLA-4 low (n = 6) and high-risk (n = 6, Unpaired t-test, p = .0298) patients as well as CD38 low (n = 5) and high-risk patients (n = 7, Unpaired t-test, p = .0278). Right. Difference in Ki-67 expression in CLL cells from patients with a combined low risk (-/-, VLA-4-/CD38-, n = 4), with discordant VLA-4 and CD38 risk (+/− −/+, n = 3), and VLA-4+/CD38+ (+/+, n = 5, ANOVA, One-way analysis of variance, p = .0233, Bonferroni's multiple comparision test). (B) Representative plot of CLL cells (CD19+/CD5+) analysed for their VLA-4 expression. VLA-4 negative and VLA-4 positive CLL cells were gated and further analyzed for their Ki-67 expression. (C) Representative plots for CD38 analysis. Percentage of Ki-67+ CLL cells in (A, right) VLA-4 negative and VLA-4 positive subclones within individual patients (n = 12, Paired t-test, p = .0275) or in (B, right) CD38 negative and CD38 positive subclones (n = 12, Paired t-test, p = .0048). neg, negative subclones; pos, positive subclones. *, P

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques: Expressing, Staining, Flow Cytometry

    VLA-4 and CD38 high-risk CLL samples show higher spontaneous apoptosis rates. PBMCs from different CLL patients were cultured in vitro for 48 hours. Viability of CLL cells was analyzed by flow cytometry after 48 hours. Difference in viability of samples from (A) VLA-4 low (n = 18) and high-risk (n = 10; Unpaired t-test, p = .0004) patients and (B) and of CD38 low (n = 19) and high-risk (n = 9; Unpaired t-test, p = .0005) patient samples. (C) Difference in viabilty after 48 hours of cells from patients with a combined low risk (−/−, VLA-4-/CD38-, n = 17), with discordant VLA-4 and CD38 risk (+/−−/+, n = 3), and with combined high risk (+/+, VLA-4+/CD38+, n = 8, ANOVA, One-way analysis of variance, p = .0007, Bonferroni's multiple comparison test). ***, P

    Journal: PLoS ONE

    Article Title: Differential Bone Marrow Homing Capacity of VLA-4 and CD38 High Expressing Chronic Lymphocytic Leukemia Cells

    doi: 10.1371/journal.pone.0023758

    Figure Lengend Snippet: VLA-4 and CD38 high-risk CLL samples show higher spontaneous apoptosis rates. PBMCs from different CLL patients were cultured in vitro for 48 hours. Viability of CLL cells was analyzed by flow cytometry after 48 hours. Difference in viability of samples from (A) VLA-4 low (n = 18) and high-risk (n = 10; Unpaired t-test, p = .0004) patients and (B) and of CD38 low (n = 19) and high-risk (n = 9; Unpaired t-test, p = .0005) patient samples. (C) Difference in viabilty after 48 hours of cells from patients with a combined low risk (−/−, VLA-4-/CD38-, n = 17), with discordant VLA-4 and CD38 risk (+/−−/+, n = 3), and with combined high risk (+/+, VLA-4+/CD38+, n = 8, ANOVA, One-way analysis of variance, p = .0007, Bonferroni's multiple comparison test). ***, P

    Article Snippet: Where indicated, PBMCs were pretreated with 0.3 µg/ml anti-VLA-4 antibodies or isotype control (BD) for 30 minutes prior addition to M2-10B4 cells.

    Techniques: Cell Culture, In Vitro, Flow Cytometry, Cytometry

    Treg transmigration through lymphatic endothelium depends on VCAM-1 in vitro and in vivo . ( a ) SVEC4-10 or mouse skin LEC: grey histograms, isotype control; blue: LTβR. Representative of 3 experiments. ( b ) SVEC4-10 cultured 48 h, with or without agonistic 1 μg ml −1 anti-LTβR last 24 h, stained for indicated molecules. Isotype control, black histograms; indicated antibody, blue (control-treated) and red (anti-LTβR-treated). Representative of two experiments. ( c , d ) Aza Treg migrated across iSVEC4-10 to CCL19. SVEC4-10 (anti-ICAM-1 and anti-VCAM-1) or Treg (anti-LFA-1, anti-VLA-4, MOPC21 and LTβRIg) pretreated as indicated. Migration normalized to control. Six transwells from two experiments in c and nine transwells from three experiments in d . ( e ) Footpad migration. CFSE-labelled WT nTreg or naive CD4+ non-Treg co-injected with Rat IgG2a or anti-VCAM-1 or pretreated with Rat IgG2b or anti-VLA-4. Per cent CFSE+ cells of popliteal LN CD4 T cells shown. Treg: 5–8 mice from 3 experiments; non-Treg: 7–10 mice from 3 experiments. ( f – i ) Aza Treg transwell migration across iSVEC4-10 ( f , g ) and primary mouse iLEC ( h , i ). Under static conditions ( f , h ) and with fluid flow ( g , i ). T cells (MOPC21 and LTβRIg) or endothelial cells (NIKi and anti-VCAM-1) treated as indicated. White numerals indicate % decline compared with MOPC21. Migration normalized to MOPC21. Results from nine transwells per condition from three experiments in f and g , and eight transwells from two experiments in h and i . ( j , k ) Transwell migration of human T cells across human skin iLEC to murine CCL19. T cells treated with MOPC21 or murine LTβRIg. ( j ) Results by well, 13 per condition from 4 experiments, red bars denote mean. ( k ) Results by human T-cell donor, control paired with LTβRIg. ( l ) Summary of Treg movement in ears during 30 min of image acquisition. In all, 563 Rat IgG2a- and 511 anti-VCAM-1-treated cells tracked using Volocity 6.1.1. Results from 2–3 ears per condition, 1 × 20 field per ear, from 2 experiments. Box and whiskers plot showing minimum, maximum, mean (+), median (bar), 25th and 75th percentiles. * P

    Journal: Nature Communications

    Article Title: Treg engage lymphotoxin beta receptor for afferent lymphatic transendothelial migration

    doi: 10.1038/ncomms12021

    Figure Lengend Snippet: Treg transmigration through lymphatic endothelium depends on VCAM-1 in vitro and in vivo . ( a ) SVEC4-10 or mouse skin LEC: grey histograms, isotype control; blue: LTβR. Representative of 3 experiments. ( b ) SVEC4-10 cultured 48 h, with or without agonistic 1 μg ml −1 anti-LTβR last 24 h, stained for indicated molecules. Isotype control, black histograms; indicated antibody, blue (control-treated) and red (anti-LTβR-treated). Representative of two experiments. ( c , d ) Aza Treg migrated across iSVEC4-10 to CCL19. SVEC4-10 (anti-ICAM-1 and anti-VCAM-1) or Treg (anti-LFA-1, anti-VLA-4, MOPC21 and LTβRIg) pretreated as indicated. Migration normalized to control. Six transwells from two experiments in c and nine transwells from three experiments in d . ( e ) Footpad migration. CFSE-labelled WT nTreg or naive CD4+ non-Treg co-injected with Rat IgG2a or anti-VCAM-1 or pretreated with Rat IgG2b or anti-VLA-4. Per cent CFSE+ cells of popliteal LN CD4 T cells shown. Treg: 5–8 mice from 3 experiments; non-Treg: 7–10 mice from 3 experiments. ( f – i ) Aza Treg transwell migration across iSVEC4-10 ( f , g ) and primary mouse iLEC ( h , i ). Under static conditions ( f , h ) and with fluid flow ( g , i ). T cells (MOPC21 and LTβRIg) or endothelial cells (NIKi and anti-VCAM-1) treated as indicated. White numerals indicate % decline compared with MOPC21. Migration normalized to MOPC21. Results from nine transwells per condition from three experiments in f and g , and eight transwells from two experiments in h and i . ( j , k ) Transwell migration of human T cells across human skin iLEC to murine CCL19. T cells treated with MOPC21 or murine LTβRIg. ( j ) Results by well, 13 per condition from 4 experiments, red bars denote mean. ( k ) Results by human T-cell donor, control paired with LTβRIg. ( l ) Summary of Treg movement in ears during 30 min of image acquisition. In all, 563 Rat IgG2a- and 511 anti-VCAM-1-treated cells tracked using Volocity 6.1.1. Results from 2–3 ears per condition, 1 × 20 field per ear, from 2 experiments. Box and whiskers plot showing minimum, maximum, mean (+), median (bar), 25th and 75th percentiles. * P

    Article Snippet: In other experiments T cells were pretreated with NA/LE grade anti-VLA-4 (clone R1-2, BD, 553153), NA/LE grade Rat IgG2b (clone R35-38, BD,555845) at 10 μg ml−1 for 30 min at 4 °C and washed 3 × with PBS before injection.

    Techniques: Transmigration Assay, In Vitro, In Vivo, Cell Culture, Staining, Migration, Injection, Mouse Assay, Flow Cytometry