Structured Review

Becton Dickinson fluorescence activated cell sorting analysis
Characterization of surface markers on M0, M1-, and M2-polarized macrophages following Toll-like receptor (TLR) ligand exposure and activation. For phenotypical <t>analysis,</t> M0 (ex vivo monocytes), M1-like (GM-CSF-differentiated), and M2-like (M-CSF-differentiated) macrophages derived from peripheral blood of healthy donors (HD) or patients with rheumatoid arthritis (RA) were stained for <t>fluorescence-activated</t> <t>cell</t> <t>sorting</t> analysis with fluorescently labeled antibodies CD14-allophycocyanin-cyanine 7 (APC-Cy7), CD163-fluorescein isothiocyanate (FITC), CD206-BV421, CD86-phycoerythrin (PE), and CD80-FITC. a Comparison of surface marker expression on freshly isolated M0- versus M1- versus M2-differentiated macrophages from HD and patients with RA presented as the percentage of positively stained cell populations. b Quality of surface marker expression in M1- versus M2-differentiated macrophages from HD and patients with RA was analyzed by mean fluorescence intensity (MFI) and presented as a box plot ( upper panel ) and with representative histograms ( lower panel ; light gray area = unstained cells, dark full line = M1, dotted line = M2). MFI was calculated as ΔMFI = MFI specific surface marker − MFI corresponding unstained control and normalized to the basal MFI of unstained control cells. c Effect of TLR or interferon (IFN)-γ/lipopolysaccharide (LPS) stimulation on surface marker expression in M1- or M2-differentiated macrophages from HD compared with patients with RA presented as percentage of positively stained cell populations. n = 6, * p
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1) Product Images from "TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype"

Article Title: TLR2 stimulation impairs anti-inflammatory activity of M2-like macrophages, generating a chimeric M1/M2 phenotype

Journal: Arthritis Research & Therapy

doi: 10.1186/s13075-017-1447-1

Characterization of surface markers on M0, M1-, and M2-polarized macrophages following Toll-like receptor (TLR) ligand exposure and activation. For phenotypical analysis, M0 (ex vivo monocytes), M1-like (GM-CSF-differentiated), and M2-like (M-CSF-differentiated) macrophages derived from peripheral blood of healthy donors (HD) or patients with rheumatoid arthritis (RA) were stained for fluorescence-activated cell sorting analysis with fluorescently labeled antibodies CD14-allophycocyanin-cyanine 7 (APC-Cy7), CD163-fluorescein isothiocyanate (FITC), CD206-BV421, CD86-phycoerythrin (PE), and CD80-FITC. a Comparison of surface marker expression on freshly isolated M0- versus M1- versus M2-differentiated macrophages from HD and patients with RA presented as the percentage of positively stained cell populations. b Quality of surface marker expression in M1- versus M2-differentiated macrophages from HD and patients with RA was analyzed by mean fluorescence intensity (MFI) and presented as a box plot ( upper panel ) and with representative histograms ( lower panel ; light gray area = unstained cells, dark full line = M1, dotted line = M2). MFI was calculated as ΔMFI = MFI specific surface marker − MFI corresponding unstained control and normalized to the basal MFI of unstained control cells. c Effect of TLR or interferon (IFN)-γ/lipopolysaccharide (LPS) stimulation on surface marker expression in M1- or M2-differentiated macrophages from HD compared with patients with RA presented as percentage of positively stained cell populations. n = 6, * p
Figure Legend Snippet: Characterization of surface markers on M0, M1-, and M2-polarized macrophages following Toll-like receptor (TLR) ligand exposure and activation. For phenotypical analysis, M0 (ex vivo monocytes), M1-like (GM-CSF-differentiated), and M2-like (M-CSF-differentiated) macrophages derived from peripheral blood of healthy donors (HD) or patients with rheumatoid arthritis (RA) were stained for fluorescence-activated cell sorting analysis with fluorescently labeled antibodies CD14-allophycocyanin-cyanine 7 (APC-Cy7), CD163-fluorescein isothiocyanate (FITC), CD206-BV421, CD86-phycoerythrin (PE), and CD80-FITC. a Comparison of surface marker expression on freshly isolated M0- versus M1- versus M2-differentiated macrophages from HD and patients with RA presented as the percentage of positively stained cell populations. b Quality of surface marker expression in M1- versus M2-differentiated macrophages from HD and patients with RA was analyzed by mean fluorescence intensity (MFI) and presented as a box plot ( upper panel ) and with representative histograms ( lower panel ; light gray area = unstained cells, dark full line = M1, dotted line = M2). MFI was calculated as ΔMFI = MFI specific surface marker − MFI corresponding unstained control and normalized to the basal MFI of unstained control cells. c Effect of TLR or interferon (IFN)-γ/lipopolysaccharide (LPS) stimulation on surface marker expression in M1- or M2-differentiated macrophages from HD compared with patients with RA presented as percentage of positively stained cell populations. n = 6, * p

Techniques Used: Activation Assay, Ex Vivo, Derivative Assay, Staining, Fluorescence, FACS, Labeling, Marker, Expressing, Isolation

2) Product Images from "Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias"

Article Title: Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias

Journal: Turkish Journal of Hematology

doi: 10.4274/tjh.2018.0022

Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls. a) Coexpression of CD14+/HLA-DR+dim. b) Coexpression of CD14+/CD124+, both representing the average of myeloid-derived suppressor cell (MDSC) percentage identified in the peripheral blood of each cohort. c) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by MDSCs in monoclonal gammopathy of unknown significance, multiple myeloma, and plasma cell leukemia patients. MM: Multiple myeloma, MGUS: monoclonal gammopathy of unknown significance, MDSC: Myeloid-derived suppressor cell, LAP: latency-associated peptide, WM: Waldenström’s macroglobulinemia.
Figure Legend Snippet: Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls. a) Coexpression of CD14+/HLA-DR+dim. b) Coexpression of CD14+/CD124+, both representing the average of myeloid-derived suppressor cell (MDSC) percentage identified in the peripheral blood of each cohort. c) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by MDSCs in monoclonal gammopathy of unknown significance, multiple myeloma, and plasma cell leukemia patients. MM: Multiple myeloma, MGUS: monoclonal gammopathy of unknown significance, MDSC: Myeloid-derived suppressor cell, LAP: latency-associated peptide, WM: Waldenström’s macroglobulinemia.

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

Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls for the expression of latency-associated peptide (LAP) on monocytes. a) Coexpression of CD14+/ LAP+. Results represent the average percentage identified in the blood of each cohort. b) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by monocytes/LAP+ cells in a healthy control and a multiple myeloma patient. LAP: Latency-associated peptide.
Figure Legend Snippet: Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls for the expression of latency-associated peptide (LAP) on monocytes. a) Coexpression of CD14+/ LAP+. Results represent the average percentage identified in the blood of each cohort. b) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by monocytes/LAP+ cells in a healthy control and a multiple myeloma patient. LAP: Latency-associated peptide.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Fluorescence

3) Product Images from "Induced Pluripotent Stem Cells Derived From Two Idiopathic Azoospermia Patients Display Compromised Differentiation Potential for Primordial Germ Cell Fate"

Article Title: Induced Pluripotent Stem Cells Derived From Two Idiopathic Azoospermia Patients Display Compromised Differentiation Potential for Primordial Germ Cell Fate

Journal: Frontiers in Cell and Developmental Biology

doi: 10.3389/fcell.2020.00432

Specification of PGCLCs from idiopathic NOA patient-specific iPSCs. (A) Schematic protocol for hPGCLCs specification from idiopathic NOA patient-specific iPSCs and images of hiPSCs, iMeLCs, and floating embryoids containing hPGCLCs. (B) (Top)Fluorescence-activated cell sorting analysis by EpCAM and INTEGRINα6 expression of day 4 embryoids differentiated from NOA iPSCs and normal hiPSCs. P2 and P3 gates (boxed areas) indicate EpCAM/INTEGRINa6-high and -low/no cells, respectively. The percentages of cells in the P2 and P3 gates are shown. (Bottom) Fluorescence-activated cell sorting analysis by c-KIT and CD38 of the two populations on the top classified by EpCAM and INTEGRINa6 expression. (C) Percentage of EpCAM/INTEGRINα6 double-positive cells in days 2, 4, 6, and 8 floating embryoids determined by FACS. Error bars indicate mean ± SD of three independent experiments. (D) Percentage of EpCAM/INTEGRINα6 double-positive cells in day 4 embryoids determined by FACS. The experiments were performed independently for more than six times. Black central line represents the median; boxes represent the 25th and 75th percentiles, and whiskers represent the maximum and minimum. Comparisons were conducted using Wilcoxon signed-ranks test. Asterisk indicates statistically significant differences ( P
Figure Legend Snippet: Specification of PGCLCs from idiopathic NOA patient-specific iPSCs. (A) Schematic protocol for hPGCLCs specification from idiopathic NOA patient-specific iPSCs and images of hiPSCs, iMeLCs, and floating embryoids containing hPGCLCs. (B) (Top)Fluorescence-activated cell sorting analysis by EpCAM and INTEGRINα6 expression of day 4 embryoids differentiated from NOA iPSCs and normal hiPSCs. P2 and P3 gates (boxed areas) indicate EpCAM/INTEGRINa6-high and -low/no cells, respectively. The percentages of cells in the P2 and P3 gates are shown. (Bottom) Fluorescence-activated cell sorting analysis by c-KIT and CD38 of the two populations on the top classified by EpCAM and INTEGRINa6 expression. (C) Percentage of EpCAM/INTEGRINα6 double-positive cells in days 2, 4, 6, and 8 floating embryoids determined by FACS. Error bars indicate mean ± SD of three independent experiments. (D) Percentage of EpCAM/INTEGRINα6 double-positive cells in day 4 embryoids determined by FACS. The experiments were performed independently for more than six times. Black central line represents the median; boxes represent the 25th and 75th percentiles, and whiskers represent the maximum and minimum. Comparisons were conducted using Wilcoxon signed-ranks test. Asterisk indicates statistically significant differences ( P

Techniques Used: FACS, Expressing, Fluorescence

Characterization of hiPSC lines derived from patients with idiopathic non-obstructive azoospermia and normal men. (A) Morphology of NOA 1106, NOA 1122, and normal hiPSCs. Scale bar, 200 μm. (B) All hiPSC lines express pluripotent genes, H1 ESCs as positive control, and human dermal fibroblasts as negative control. (C) NOA 1106 iPSCs show the expression of protein markers for pluripotency. Scale bar, 100 μm. (D) Fluorescence-activated cell sorting analysis for OCT4, NANOG, and SOX2 expression in NOA 1106 iPSCs. (E) In vivo and in vitro differentiation of NOA 1106 iPSCs. (Left) Hematoxylin–eosin staining of teratoma sections from NOA 1106 iPSCs shows the evidence of all three germ layers: respiratory epithelium (endoderm), cartilage (mesoderm), and pigmented cells (ectoderm). Scale bar, 100 μm. (Right) Embryoid bodies (EBs) derived from the NOA 1106 iPSCs in vitro . Scale bar, 200 μm. (F) NOA 1106 iPSCs exhibit normal karyotype in G-band analysis. (G) The differentiated cells from the EBs formed by NOA 1106 iPSCs express genes representative of all three germ layers. See also Supplementary Figure S1 .
Figure Legend Snippet: Characterization of hiPSC lines derived from patients with idiopathic non-obstructive azoospermia and normal men. (A) Morphology of NOA 1106, NOA 1122, and normal hiPSCs. Scale bar, 200 μm. (B) All hiPSC lines express pluripotent genes, H1 ESCs as positive control, and human dermal fibroblasts as negative control. (C) NOA 1106 iPSCs show the expression of protein markers for pluripotency. Scale bar, 100 μm. (D) Fluorescence-activated cell sorting analysis for OCT4, NANOG, and SOX2 expression in NOA 1106 iPSCs. (E) In vivo and in vitro differentiation of NOA 1106 iPSCs. (Left) Hematoxylin–eosin staining of teratoma sections from NOA 1106 iPSCs shows the evidence of all three germ layers: respiratory epithelium (endoderm), cartilage (mesoderm), and pigmented cells (ectoderm). Scale bar, 100 μm. (Right) Embryoid bodies (EBs) derived from the NOA 1106 iPSCs in vitro . Scale bar, 200 μm. (F) NOA 1106 iPSCs exhibit normal karyotype in G-band analysis. (G) The differentiated cells from the EBs formed by NOA 1106 iPSCs express genes representative of all three germ layers. See also Supplementary Figure S1 .

Techniques Used: Derivative Assay, Positive Control, Negative Control, Expressing, Fluorescence, FACS, In Vivo, In Vitro, Staining

Differentially expressed gene analysis between PGCLCs derived from NOA and normal hiPSCs. (A) Heat map of gene expression of key PGC-associated genes (early and late stage) and of pluripotency, mesoderm, endoderm, ectoderm, and ICM genes. (B) Numbers of genes upregulated or downregulated at key stages during PGCLC specification of NOA 1106 iPSCs (left) and NOA 1122 iPSCs (right) compared with that of normal iPSCs. (C) Volcano plot of the DEGs in PGCLCs derived from NOA 1106 iPSCs (left) and NOA 1122 iPSCs (right) compared with PGCLCs derived from normal iPSCs. The red dots indicate genes upregulated, and the green dots indicate genes downregulated. (D) Enriched GO terms in the upregulated genes of PGCLCs derived from NOA 1106 iPSCs (left) and NOA 1122 iPSCs (right) compared with PGCLCs derived from normal iPSCs. Primordial germ cell–like cells, the EpCAM, and INTEGRINα6 double-positive cells in day 4 embryoids. (E) Fluorescence-activated cell sorting analysis by annexin V and PI staining for day 4 embryoids differentiated from NOA iPSCs and normal hiPSCs. The percentages of cells in the four quadrants are shown. (F) Apoptotic rates of day 4 embryoids differentiated from NOA iPSCs and normal hiPSCs. Error bars indicate mean ± SD of three independent experiments. Comparisons were conducted using ANOVA. Asterisk indicated statistically significant differences ( P
Figure Legend Snippet: Differentially expressed gene analysis between PGCLCs derived from NOA and normal hiPSCs. (A) Heat map of gene expression of key PGC-associated genes (early and late stage) and of pluripotency, mesoderm, endoderm, ectoderm, and ICM genes. (B) Numbers of genes upregulated or downregulated at key stages during PGCLC specification of NOA 1106 iPSCs (left) and NOA 1122 iPSCs (right) compared with that of normal iPSCs. (C) Volcano plot of the DEGs in PGCLCs derived from NOA 1106 iPSCs (left) and NOA 1122 iPSCs (right) compared with PGCLCs derived from normal iPSCs. The red dots indicate genes upregulated, and the green dots indicate genes downregulated. (D) Enriched GO terms in the upregulated genes of PGCLCs derived from NOA 1106 iPSCs (left) and NOA 1122 iPSCs (right) compared with PGCLCs derived from normal iPSCs. Primordial germ cell–like cells, the EpCAM, and INTEGRINα6 double-positive cells in day 4 embryoids. (E) Fluorescence-activated cell sorting analysis by annexin V and PI staining for day 4 embryoids differentiated from NOA iPSCs and normal hiPSCs. The percentages of cells in the four quadrants are shown. (F) Apoptotic rates of day 4 embryoids differentiated from NOA iPSCs and normal hiPSCs. Error bars indicate mean ± SD of three independent experiments. Comparisons were conducted using ANOVA. Asterisk indicated statistically significant differences ( P

Techniques Used: Derivative Assay, Expressing, Pyrolysis Gas Chromatography, Fluorescence, FACS, Staining

4) Product Images from "Suberoylanilide hydroxamic acid induces apoptosis and sub-G1 arrest of 320 HSR colon cancer cells"

Article Title: Suberoylanilide hydroxamic acid induces apoptosis and sub-G1 arrest of 320 HSR colon cancer cells

Journal: Journal of Biomedical Science

doi: 10.1186/1423-0127-17-76

SAHA induces apoptosis in colon cancer cell lines . 320 HSR cells were stained with Annexin V (FITC) and propidium iodide (PI) after treatment with SAHA. Fluorescence-activated cell sorting analysis of 320 HSR cancer cell line at 48 h following treatment with 0, 1, 2.5, and 5 μM SAHA (A, B, C, D, respectively). Percentages represent Annexin V-positive/PI-negative (early apoptotic) and Annexin V-positive/PI-positive cells (apoptotic).
Figure Legend Snippet: SAHA induces apoptosis in colon cancer cell lines . 320 HSR cells were stained with Annexin V (FITC) and propidium iodide (PI) after treatment with SAHA. Fluorescence-activated cell sorting analysis of 320 HSR cancer cell line at 48 h following treatment with 0, 1, 2.5, and 5 μM SAHA (A, B, C, D, respectively). Percentages represent Annexin V-positive/PI-negative (early apoptotic) and Annexin V-positive/PI-positive cells (apoptotic).

Techniques Used: Staining, Fluorescence, FACS

Fluorescence activated cell sorting (FACS) analysis revealed SAHA-induced sub-G1 arrest in 320HSR cells . Cells were harvested 48 h after stimulation in the absence or presence of SAHA (0.1 μM to 5 μM). Intracellular PI fluorescence intensities of cells are presented in the upper panels. The percentage of cells in the G0/G1 phase was significantly inhibited by SAHA treatment after 24 or 48 h. The percentage of cells in the sub-G1 phase was significantly increased in response to SAHA treatment.
Figure Legend Snippet: Fluorescence activated cell sorting (FACS) analysis revealed SAHA-induced sub-G1 arrest in 320HSR cells . Cells were harvested 48 h after stimulation in the absence or presence of SAHA (0.1 μM to 5 μM). Intracellular PI fluorescence intensities of cells are presented in the upper panels. The percentage of cells in the G0/G1 phase was significantly inhibited by SAHA treatment after 24 or 48 h. The percentage of cells in the sub-G1 phase was significantly increased in response to SAHA treatment.

Techniques Used: Fluorescence, FACS

5) Product Images from "Preimmunization of donor lymphocytes enhances antitumor immunity of autologous hematopoietic stem cell transplantation"

Article Title: Preimmunization of donor lymphocytes enhances antitumor immunity of autologous hematopoietic stem cell transplantation

Journal: Cancer Medicine

doi: 10.1002/cam4.117

Syngeneic HSCT suppressed the growth of subcutaneous tumors. (A) Growth suppression of subcutaneous tumors in syngeneic HSCT mice. The BALB/c mice received a lethal dose of irradiation, followed by a infusion of BM and splenic T cells derived from BALB/c mice. CT26 cells were inoculated into right legs (number of animals per each group: n = 7). Tumor volumes were measured at the indicated days. (B) The increase of IFN-γ-positive cells in response to stimulation of CT26 cells by an ELISpot assay. Two weeks after HSCT, splenocytes were isolated from HSCT mice, and cocultured with CT26 cells or control lymphocytes ( n = 3). (C) The increase of tumor-specific CD8 + T cells in HSCT mice. The splenocytes were isolated 2 weeks after HSCT, and CT26-specific AH-1-tetramer-positive cells were analyzed by flow cytometry ( n = 4). CD8 + lymphocyte regions in FACS plots were gated and developed spots in two dimensions, and the ratio of tetramer + CD8 + cells per total CD8 + cells was calculated. (D) The increase of proliferating activity of CD3 + cells in HSCT mice. HSCT was performed 2 weeks after tumor inoculation, and the proliferation of CD3 + T cells was analyzed during 3–5 weeks after tumor inoculation (1–3 weeks after HSCT). The splenocytes were isolated and 5 × 10 6 of carboxyfluorescein succinimidyl ester (CSFE)-labeled splenocytes were cultured with CD11c + cells in CD3-coated 24-well plates. After 48 h, the proliferating fraction of CD3 + cells was evaluated with anti-CD3 + antibody (BD Biosciences) by flow cytometry ( n = 3). The proliferating cells were defined as a single or more round of division. HSCT, hematopoietic stem cell transplantation; BM, bone marrow; IFN, interferon; FACS, fluorescence-activated cell sorting.
Figure Legend Snippet: Syngeneic HSCT suppressed the growth of subcutaneous tumors. (A) Growth suppression of subcutaneous tumors in syngeneic HSCT mice. The BALB/c mice received a lethal dose of irradiation, followed by a infusion of BM and splenic T cells derived from BALB/c mice. CT26 cells were inoculated into right legs (number of animals per each group: n = 7). Tumor volumes were measured at the indicated days. (B) The increase of IFN-γ-positive cells in response to stimulation of CT26 cells by an ELISpot assay. Two weeks after HSCT, splenocytes were isolated from HSCT mice, and cocultured with CT26 cells or control lymphocytes ( n = 3). (C) The increase of tumor-specific CD8 + T cells in HSCT mice. The splenocytes were isolated 2 weeks after HSCT, and CT26-specific AH-1-tetramer-positive cells were analyzed by flow cytometry ( n = 4). CD8 + lymphocyte regions in FACS plots were gated and developed spots in two dimensions, and the ratio of tetramer + CD8 + cells per total CD8 + cells was calculated. (D) The increase of proliferating activity of CD3 + cells in HSCT mice. HSCT was performed 2 weeks after tumor inoculation, and the proliferation of CD3 + T cells was analyzed during 3–5 weeks after tumor inoculation (1–3 weeks after HSCT). The splenocytes were isolated and 5 × 10 6 of carboxyfluorescein succinimidyl ester (CSFE)-labeled splenocytes were cultured with CD11c + cells in CD3-coated 24-well plates. After 48 h, the proliferating fraction of CD3 + cells was evaluated with anti-CD3 + antibody (BD Biosciences) by flow cytometry ( n = 3). The proliferating cells were defined as a single or more round of division. HSCT, hematopoietic stem cell transplantation; BM, bone marrow; IFN, interferon; FACS, fluorescence-activated cell sorting.

Techniques Used: Mouse Assay, Irradiation, Derivative Assay, Enzyme-linked Immunospot, Isolation, Flow Cytometry, Cytometry, FACS, Activity Assay, Labeling, Cell Culture, Transplantation Assay, Fluorescence

6) Product Images from "Role of Macrophage Socs3 in the Pathogenesis of Aortic Dissection"

Article Title: Role of Macrophage Socs3 in the Pathogenesis of Aortic Dissection

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

doi: 10.1161/JAHA.117.007389

Functional differentiation of macrophages in the aorta. Results of the FACS analysis are shown for the functional differentiation of macrophages in aortae from WT (left panel) and mS ocs3‐ KO (right panel) mice treated with Ca+Ang II to induce focal medial disruption. Macrophages were defined as the CD 11b hi population after gating for 7‐ AAD − live cells, CD 45 + hematopoietic cells, and Ly6G − non‐neutrophils. Macrophage differentiation status was defined as Ly6C hi tissue‐destructive and Ly6C lo reparative macrophages. Representative scattergrams for WT and mS ocs3‐ KO mice are shown for at least 3 mice for each experimental group. Two additional independent sets of experiments showed similar results with regard to the ratio of tissue‐destructive and reparative macrophages. 7‐AAD indicates 7‐aminoactinomycin D; AngII, angiotensin II; FACS, fluorescence‐activated cell sorting; WT, wild type.
Figure Legend Snippet: Functional differentiation of macrophages in the aorta. Results of the FACS analysis are shown for the functional differentiation of macrophages in aortae from WT (left panel) and mS ocs3‐ KO (right panel) mice treated with Ca+Ang II to induce focal medial disruption. Macrophages were defined as the CD 11b hi population after gating for 7‐ AAD − live cells, CD 45 + hematopoietic cells, and Ly6G − non‐neutrophils. Macrophage differentiation status was defined as Ly6C hi tissue‐destructive and Ly6C lo reparative macrophages. Representative scattergrams for WT and mS ocs3‐ KO mice are shown for at least 3 mice for each experimental group. Two additional independent sets of experiments showed similar results with regard to the ratio of tissue‐destructive and reparative macrophages. 7‐AAD indicates 7‐aminoactinomycin D; AngII, angiotensin II; FACS, fluorescence‐activated cell sorting; WT, wild type.

Techniques Used: Functional Assay, FACS, Mass Spectrometry, Mouse Assay, Fluorescence

7) Product Images from "Rational cotargeting of HDAC6 and BET proteins yields synergistic antimyeloma activity"

Article Title: Rational cotargeting of HDAC6 and BET proteins yields synergistic antimyeloma activity

Journal: Blood Advances

doi: 10.1182/bloodadvances.2018026484

JQ1 reduces the cell viability of MM cells, modulates c-MYC and p21 expression, and induces apoptosis. (A) JQ1 decreases MM cell viability. A panel of MM cell lines was treated with the indicated concentrations of JQ1 for 72 hours. Cell viability was measured by MTT assay. (B) Quantitative RT-PCR (qRT-PCR) analysis of c-MYC and CDKN1A levels. MM cells were treated with 500 nM JQ1 for 24 hours, and gene expression was measured by qRT-PCR. (C) JQ1 treatment decreases c-MYC protein expression and induces p21. RPMI-8226, LP-1, and OPM-2 MM cells were treated with the indicated concentrations of JQ1 for 24 hours (left) or 500 nM JQ1 (right) for the indicated times. Protein expression was determined by immunoblotting. (D) JQ1 stimulates apoptosis in MM cell lines. LP-1 and OPM-2 MM cells were treated with the indicated concentrations of JQ1 for 48 hours. Apoptosis was determined by propidium iodide fluorescence-activated cell sorting (PI-FACS) analysis. Data are shown as mean ± standard deviation (SD); n = 3. *Indicates a significant difference from controls. P
Figure Legend Snippet: JQ1 reduces the cell viability of MM cells, modulates c-MYC and p21 expression, and induces apoptosis. (A) JQ1 decreases MM cell viability. A panel of MM cell lines was treated with the indicated concentrations of JQ1 for 72 hours. Cell viability was measured by MTT assay. (B) Quantitative RT-PCR (qRT-PCR) analysis of c-MYC and CDKN1A levels. MM cells were treated with 500 nM JQ1 for 24 hours, and gene expression was measured by qRT-PCR. (C) JQ1 treatment decreases c-MYC protein expression and induces p21. RPMI-8226, LP-1, and OPM-2 MM cells were treated with the indicated concentrations of JQ1 for 24 hours (left) or 500 nM JQ1 (right) for the indicated times. Protein expression was determined by immunoblotting. (D) JQ1 stimulates apoptosis in MM cell lines. LP-1 and OPM-2 MM cells were treated with the indicated concentrations of JQ1 for 48 hours. Apoptosis was determined by propidium iodide fluorescence-activated cell sorting (PI-FACS) analysis. Data are shown as mean ± standard deviation (SD); n = 3. *Indicates a significant difference from controls. P

Techniques Used: Expressing, MTT Assay, Quantitative RT-PCR, Fluorescence, FACS, Standard Deviation

8) Product Images from "The organotelluride catalyst LAB027 prevents colon cancer growth in the mice"

Article Title: The organotelluride catalyst LAB027 prevents colon cancer growth in the mice

Journal: Cell Death & Disease

doi: 10.1038/cddis.2011.73

Pro-apoptotic effects of LAB027 ( a ). HT29 cells were treated or not with increasing concentrations of LAB027 for 24 h. Expressions of cleaved caspase-8, pro-caspase-3 and cleaved caspase-3, Bax, Bad, Bcl-xL, Bcl-2 and ATG8 proteins were analyzed by western blot. Proliferation rates ( b ) of CT26, HT29 and NIH3T3 cells incubated with various concentrations of LAB027 alone or associated with either 40 μ M DEVD-FMK (caspase-3 inhibitor) or 40 μ M IETD-FMK (caspase-8 inhibitor) 40 μ M ZVAD-FMK (broad-spectrum caspase inhibitor). Results are means±S.E.M. of three independent experiments. ( c ) Immunofluorescence microscopy of HT29 cells stained with acridine orange and treated for 24 h or not with 4 μ M LAB027. An increased number of cells with stained acidic vesicular organelles (orange fluorescence) in 4 μ M LAB027 treated cells was observed. ( d ) Fluorescence-activated cell sorting analysis of cell death. Apoptosis/necrosis was analyzed by flow cytometry using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO-1 and Propidium iodide on HT29 treated (right panel) or not (left panel) with 4 μ M LAB027 for 24 h. Necrotic cells are PI positive and YO-PRO-1 positive or negative. Apoptotic cells are PI negative and YO-PRO-1 positive. Viable cells are negative for both dyes. One representative experiment of three is shown. ( e ) Kinetic apoptosis/necrosis evolution in HT29 cells treated with 4 μ M LAB027 alone or with oxaliplatin 0.5 μ M
Figure Legend Snippet: Pro-apoptotic effects of LAB027 ( a ). HT29 cells were treated or not with increasing concentrations of LAB027 for 24 h. Expressions of cleaved caspase-8, pro-caspase-3 and cleaved caspase-3, Bax, Bad, Bcl-xL, Bcl-2 and ATG8 proteins were analyzed by western blot. Proliferation rates ( b ) of CT26, HT29 and NIH3T3 cells incubated with various concentrations of LAB027 alone or associated with either 40 μ M DEVD-FMK (caspase-3 inhibitor) or 40 μ M IETD-FMK (caspase-8 inhibitor) 40 μ M ZVAD-FMK (broad-spectrum caspase inhibitor). Results are means±S.E.M. of three independent experiments. ( c ) Immunofluorescence microscopy of HT29 cells stained with acridine orange and treated for 24 h or not with 4 μ M LAB027. An increased number of cells with stained acidic vesicular organelles (orange fluorescence) in 4 μ M LAB027 treated cells was observed. ( d ) Fluorescence-activated cell sorting analysis of cell death. Apoptosis/necrosis was analyzed by flow cytometry using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO-1 and Propidium iodide on HT29 treated (right panel) or not (left panel) with 4 μ M LAB027 for 24 h. Necrotic cells are PI positive and YO-PRO-1 positive or negative. Apoptotic cells are PI negative and YO-PRO-1 positive. Viable cells are negative for both dyes. One representative experiment of three is shown. ( e ) Kinetic apoptosis/necrosis evolution in HT29 cells treated with 4 μ M LAB027 alone or with oxaliplatin 0.5 μ M

Techniques Used: Western Blot, Incubation, Immunofluorescence, Microscopy, Staining, Fluorescence, FACS, Flow Cytometry, Cytometry, Permeability

9) Product Images from "Indoxyl Sulfate Affects Glial Function Increasing Oxidative Stress and Neuroinflammation in Chronic Kidney Disease: Interaction between Astrocytes and Microglia"

Article Title: Indoxyl Sulfate Affects Glial Function Increasing Oxidative Stress and Neuroinflammation in Chronic Kidney Disease: Interaction between Astrocytes and Microglia

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2017.00370

Effect of IS (15–60 μM) on ROS formation (A) , evaluated by means of the probe H 2 DCF-DA, in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (15–60 μM) on nitrotyrosine formmation (B) in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (30 μM) on Nrf2 nuclear translocation in astrocytes and mixed glial cells (C) . Nuclear translocation of Nrf2 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on HO-1 expression (D) in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity ( n = 9). ∘∘∘ , ∘∘ , and °denote P
Figure Legend Snippet: Effect of IS (15–60 μM) on ROS formation (A) , evaluated by means of the probe H 2 DCF-DA, in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (15–60 μM) on nitrotyrosine formmation (B) in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (30 μM) on Nrf2 nuclear translocation in astrocytes and mixed glial cells (C) . Nuclear translocation of Nrf2 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on HO-1 expression (D) in astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity ( n = 9). ∘∘∘ , ∘∘ , and °denote P

Techniques Used: Fluorescence, FACS, Software, Translocation Assay, Immunofluorescence, Confocal Microscopy, Confocal Laser Scanning Microscopy, Expressing

Effect of IS (15–60 μM) on iNOS (A) , COX-2 (B) expression by astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity ( n = 9). Effect of IS (15–60 μM) TNF-α (C) and IL-6 (D) release by astrocytes and mixed glial cells ( n = 9). Cyokine release was assessed by ELISA assay and expressed as pg/ml ( n = 9). ∘∘∘ , ∘∘ , and °denote P
Figure Legend Snippet: Effect of IS (15–60 μM) on iNOS (A) , COX-2 (B) expression by astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity ( n = 9). Effect of IS (15–60 μM) TNF-α (C) and IL-6 (D) release by astrocytes and mixed glial cells ( n = 9). Cyokine release was assessed by ELISA assay and expressed as pg/ml ( n = 9). ∘∘∘ , ∘∘ , and °denote P

Techniques Used: Expressing, Fluorescence, FACS, Software, Enzyme-linked Immunosorbent Assay

Effect of IS (15–60 μM) on ROS formation (A) , evaluated by means of the probe H 2 DCF-DA, in C6 cells in presence of DPI and of NAC. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity ( n = 12). Effect of IS (30 μM) on Nrf2 nuclear translocation in C6 cells in presence of DPI and NAC (B) . Nuclear translocation of Nrf2 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of the nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on HO-1 (C) , NQO1 (D) , and SOD (E) expression in C6 cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (30 μM) on AhR nuclear translocation in presence of DPI in C6 cells (F) . Nuclear translocation of AhR was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and AhR, respectively. Analysis was performed by confocal laser scanning microscopy and values are expressed as mean fluorescence intensity ( n = 12). Effect of IS (15–60 μM) on ROS formation (G) , evaluated by means of the probe H 2 DCF-DA, in C6 cells in presence of CH-223191. Values are expressed as mean fluorescence intensity ( n = 9). ∘∘∘ , ∘∘ , and °denote P
Figure Legend Snippet: Effect of IS (15–60 μM) on ROS formation (A) , evaluated by means of the probe H 2 DCF-DA, in C6 cells in presence of DPI and of NAC. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Values are expressed as mean fluorescence intensity ( n = 12). Effect of IS (30 μM) on Nrf2 nuclear translocation in C6 cells in presence of DPI and NAC (B) . Nuclear translocation of Nrf2 was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of the nucleus (DAPI) and Nrf2, respectively. Analysis was performed by confocal laser scanning microscopy. Effect of IS (15–60 μM) on HO-1 (C) , NQO1 (D) , and SOD (E) expression in C6 cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Effect of IS (30 μM) on AhR nuclear translocation in presence of DPI in C6 cells (F) . Nuclear translocation of AhR was detected using immunofluorescence confocal microscopy. Scale bar, 10 μm. Blue and green fluorescences indicate localization of nucleus (DAPI) and AhR, respectively. Analysis was performed by confocal laser scanning microscopy and values are expressed as mean fluorescence intensity ( n = 12). Effect of IS (15–60 μM) on ROS formation (G) , evaluated by means of the probe H 2 DCF-DA, in C6 cells in presence of CH-223191. Values are expressed as mean fluorescence intensity ( n = 9). ∘∘∘ , ∘∘ , and °denote P

Techniques Used: Fluorescence, FACS, Software, Translocation Assay, Immunofluorescence, Confocal Microscopy, Confocal Laser Scanning Microscopy, Expressing

10) Product Images from "AST-120 Reduces Neuroinflammation Induced by Indoxyl Sulfate in Glial Cells"

Article Title: AST-120 Reduces Neuroinflammation Induced by Indoxyl Sulfate in Glial Cells

Journal: Journal of Clinical Medicine

doi: 10.3390/jcm7100365

Effect of IS (60, 30 and 15 μM) in inflammatory conditions, induced by LPS (1 µg/mL) + IFN (100 U/mL), on NO relase ( Panel A ), evaluated as NO 2 [µM]. Effect of IS (15–60 μM) in presence of LPS (1 µg/mL) + IFN (100 U/mL) on iNOS ( Panel B ), and COX-2 ( Panel D ) expression in primary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson), and elaborated with Cell Quest software. Panel C shows the representative fluorescence images for iNOS expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM + LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the representative fluorescence images for COX-2 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM with LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM with LPS + IFN. Effect of IS (15, 30, and 60 μM) in inflammatory conditions, induced by LPS (1 µg/mL) and IFN (100 U/mL) on TNF-α ( Panel F ) and on IL-6 ( Panel G ) release by astrocytes and mixed glial cells. Cyokine release was assessed by ELISA assay. Values are expressed as NO 2 - release, or mean fluorescence intensity or as pg/mL protein for cytokines. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°° denotes p
Figure Legend Snippet: Effect of IS (60, 30 and 15 μM) in inflammatory conditions, induced by LPS (1 µg/mL) + IFN (100 U/mL), on NO relase ( Panel A ), evaluated as NO 2 [µM]. Effect of IS (15–60 μM) in presence of LPS (1 µg/mL) + IFN (100 U/mL) on iNOS ( Panel B ), and COX-2 ( Panel D ) expression in primary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson), and elaborated with Cell Quest software. Panel C shows the representative fluorescence images for iNOS expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM + LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the representative fluorescence images for COX-2 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM with LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM with LPS + IFN. Effect of IS (15, 30, and 60 μM) in inflammatory conditions, induced by LPS (1 µg/mL) and IFN (100 U/mL) on TNF-α ( Panel F ) and on IL-6 ( Panel G ) release by astrocytes and mixed glial cells. Cyokine release was assessed by ELISA assay. Values are expressed as NO 2 - release, or mean fluorescence intensity or as pg/mL protein for cytokines. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°° denotes p

Techniques Used: Expressing, Fluorescence, FACS, Software, Enzyme-linked Immunosorbent Assay

Effect of IS (60, 30 and 15 μM) on primary CNS cells treated with LPS (1 µg/mL) + IFN (100 U/mL) on nitrotyrosine formation ( Panel A ), and on ROS release ( Panel B ), evaluated by means of the probe 2’,7’ dichlorofluorescein-diacetate (H 2 DCF-DA) and on HO-1 expression ( Panel C ) in prmary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Panel D shows the representative fluorescence images for HO-1 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM in presence of LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the effect of supernatant from IS-treated microglia on cortical and hippocampal neuronal cell viability. Values are expressed as mean fluorescence intensity or as cell citotoxicity. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°°, °° denote p
Figure Legend Snippet: Effect of IS (60, 30 and 15 μM) on primary CNS cells treated with LPS (1 µg/mL) + IFN (100 U/mL) on nitrotyrosine formation ( Panel A ), and on ROS release ( Panel B ), evaluated by means of the probe 2’,7’ dichlorofluorescein-diacetate (H 2 DCF-DA) and on HO-1 expression ( Panel C ) in prmary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Panel D shows the representative fluorescence images for HO-1 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM in presence of LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the effect of supernatant from IS-treated microglia on cortical and hippocampal neuronal cell viability. Values are expressed as mean fluorescence intensity or as cell citotoxicity. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°°, °° denote p

Techniques Used: Expressing, Fluorescence, FACS, Software

11) Product Images from "Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia"

Article Title: Vitamin C-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia

Journal: Leukemia

doi: 10.1038/leu.2017.171

Vitamin C reduces cell proliferation and modulates gene expression in IDH1R132H-expressing cells. ( a ) Mean fluorescence values from 5hmC immunofluorescence experiments±s.e.m. ( b ) A growth curve represented as mean cell count±s.d., for untreated (grey) or vitamin C-treated (orange; 0.345 m m ) IDH1 wt and IDH1 R132H cells over 9 days. ( c ) The proportion of fluorescence-activated cell sorting sorted cells with each indicated marker alone or in combination. Untreated (grey) or vitamin C-treated (orange; 72 h) IDH1 WT or IDH1 R132H cells; represented by mean proportion±s.e.m. ( d ) RNA-seq expression values, represented as log10(RPKM), for each gene in mm10 Ensembl v71 (RPKM > 0.01) in untreated ( x axis) and vitamin C-treated ( y axis) cells. Upregulated (up), downregulated (down) and unaffected genes (false discovery rate
Figure Legend Snippet: Vitamin C reduces cell proliferation and modulates gene expression in IDH1R132H-expressing cells. ( a ) Mean fluorescence values from 5hmC immunofluorescence experiments±s.e.m. ( b ) A growth curve represented as mean cell count±s.d., for untreated (grey) or vitamin C-treated (orange; 0.345 m m ) IDH1 wt and IDH1 R132H cells over 9 days. ( c ) The proportion of fluorescence-activated cell sorting sorted cells with each indicated marker alone or in combination. Untreated (grey) or vitamin C-treated (orange; 72 h) IDH1 WT or IDH1 R132H cells; represented by mean proportion±s.e.m. ( d ) RNA-seq expression values, represented as log10(RPKM), for each gene in mm10 Ensembl v71 (RPKM > 0.01) in untreated ( x axis) and vitamin C-treated ( y axis) cells. Upregulated (up), downregulated (down) and unaffected genes (false discovery rate

Techniques Used: Expressing, Fluorescence, Immunofluorescence, Cell Counting, FACS, Marker, RNA Sequencing Assay

12) Product Images from "Aldose Reductase Mediates NLRP3 Inflammasome–Initiated Innate Immune Response in Hyperglycemia-Induced Thp1 Monocytes and Male Mice"

Article Title: Aldose Reductase Mediates NLRP3 Inflammasome–Initiated Innate Immune Response in Hyperglycemia-Induced Thp1 Monocytes and Male Mice

Journal: Endocrinology

doi: 10.1210/en.2017-00294

AR inhibition suppressed HG-induced ROS production in Thp1 monocytes. (a) Thp1 cells (1 × 10 6 cells/mL) were pretreated overnight with fidarestat (10 µM), followed by incubation with HG (25 mM) for 0, 15, 30, 60, and 120 minutes. Intracellular ROS were measured using the cell-permeable dye CM-H 2 DCFDA (Invitrogen, Molecular Probes) and fluorescence images (×20 magnification) were obtained using a fluorescence microscope. (b) ROS levels were quantitated by determining the fluorescence intensity by using a fluorescence plate reader, as described in Methods. (c, d) Fluorescence-activated cell sorting analysis showing the CM-H 2 DCFDA fluorescence in Thp1 cells treated with HG with or without fidarestat for 2 hours. (c) Red, control; green, fidarestat; black (filled), HG; blue, HG plus fidarestat; black (unfilled), unstained control cells. (d) Bars show the mean fluorescence intensity. Data shown as mean ± standard deviation (n = 3). * P
Figure Legend Snippet: AR inhibition suppressed HG-induced ROS production in Thp1 monocytes. (a) Thp1 cells (1 × 10 6 cells/mL) were pretreated overnight with fidarestat (10 µM), followed by incubation with HG (25 mM) for 0, 15, 30, 60, and 120 minutes. Intracellular ROS were measured using the cell-permeable dye CM-H 2 DCFDA (Invitrogen, Molecular Probes) and fluorescence images (×20 magnification) were obtained using a fluorescence microscope. (b) ROS levels were quantitated by determining the fluorescence intensity by using a fluorescence plate reader, as described in Methods. (c, d) Fluorescence-activated cell sorting analysis showing the CM-H 2 DCFDA fluorescence in Thp1 cells treated with HG with or without fidarestat for 2 hours. (c) Red, control; green, fidarestat; black (filled), HG; blue, HG plus fidarestat; black (unfilled), unstained control cells. (d) Bars show the mean fluorescence intensity. Data shown as mean ± standard deviation (n = 3). * P

Techniques Used: Inhibition, Incubation, Fluorescence, Microscopy, FACS, Standard Deviation

13) Product Images from "Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis"

Article Title: Smac mimetic and demethylating agents synergistically trigger cell death in acute myeloid leukemia cells and overcome apoptosis resistance by inducing necroptosis

Journal: Cell Death & Disease

doi: 10.1038/cddis.2013.320

BV6 and DAC cooperate to trigger caspase activation, mitochondrial perturbations and DNA fragmentation. ( a ) MV4-11 and NB4 cells were treated for 72 h with indicated concentrations of BV6 and/or DAC (MV4-11: 600 nM BV6, 30 nM DAC; NB4: 100 nM BV6, 50 nM DAC). Apoptosis was determined by fluorescence-activated cell sorting analysis of DNA fragmentation of PI-stained nuclei. Mean and SD of three experiments performed in triplicate are shown. * P
Figure Legend Snippet: BV6 and DAC cooperate to trigger caspase activation, mitochondrial perturbations and DNA fragmentation. ( a ) MV4-11 and NB4 cells were treated for 72 h with indicated concentrations of BV6 and/or DAC (MV4-11: 600 nM BV6, 30 nM DAC; NB4: 100 nM BV6, 50 nM DAC). Apoptosis was determined by fluorescence-activated cell sorting analysis of DNA fragmentation of PI-stained nuclei. Mean and SD of three experiments performed in triplicate are shown. * P

Techniques Used: Activation Assay, Fluorescence, FACS, Staining

BV6/DAC combination treatment bypasses apoptosis resistance via a switch to necroptosis. ( a ) Cells were treated for 6 h (MV4-11) or 48 h (NB4) with BV6 and DAC (MV4-11: 600 nM BV6, 30 nM DAC; NB4: 100 nM BV6, 50 nM DAC) in the presence or absence of 20 μ M zVAD.fmk. ROS production was determined by CellROX staining and flow cytometry, and is shown as fold increase. ( b ) MV4-11 cells were treated for 6 h with BV6 and DAC (MV4-11: 600 nM BV6, 30 nM DAC) in the presence or absence of 20 μ M zVAD.fmk with or without pretreatment with 10 mM NAC for 24 h. Cell death was determined by FSC/SSC and fluorescence-activated cell sorting analysis. ( c and d ) Cells were treated for 6 h (MV4-11, Molm13) or 48 h (NB4) with BV6 and DAC (MV4-11: 600 nM BV6, 30 nM DAC; NB4: 100 nM BV6, 50 nM DAC; Molm13: 400 nM BV6, 100 nM DAC) in the presence of 20 μ M zVAD.fmk and/or 30 μ M Nec-1 ( c ), or 1 μ M NSA ( d ). Cell death was determined by FSC/SSC and FACS analysis. Mean and SD of at least three experiments performed in triplicate are shown; * P
Figure Legend Snippet: BV6/DAC combination treatment bypasses apoptosis resistance via a switch to necroptosis. ( a ) Cells were treated for 6 h (MV4-11) or 48 h (NB4) with BV6 and DAC (MV4-11: 600 nM BV6, 30 nM DAC; NB4: 100 nM BV6, 50 nM DAC) in the presence or absence of 20 μ M zVAD.fmk. ROS production was determined by CellROX staining and flow cytometry, and is shown as fold increase. ( b ) MV4-11 cells were treated for 6 h with BV6 and DAC (MV4-11: 600 nM BV6, 30 nM DAC) in the presence or absence of 20 μ M zVAD.fmk with or without pretreatment with 10 mM NAC for 24 h. Cell death was determined by FSC/SSC and fluorescence-activated cell sorting analysis. ( c and d ) Cells were treated for 6 h (MV4-11, Molm13) or 48 h (NB4) with BV6 and DAC (MV4-11: 600 nM BV6, 30 nM DAC; NB4: 100 nM BV6, 50 nM DAC; Molm13: 400 nM BV6, 100 nM DAC) in the presence of 20 μ M zVAD.fmk and/or 30 μ M Nec-1 ( c ), or 1 μ M NSA ( d ). Cell death was determined by FSC/SSC and FACS analysis. Mean and SD of at least three experiments performed in triplicate are shown; * P

Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence, FACS

14) Product Images from "Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias"

Article Title: Hierarchical Involvement of Myeloid-Derived Suppressor Cells and Monocytes Expressing Latency-Associated Peptide in Plasma Cell Dyscrasias

Journal: Turkish Journal of Hematology

doi: 10.4274/tjh.2018.0022

Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls for the expression of latency-associated peptide (LAP) on monocytes. a) Coexpression of CD14+/ LAP+. Results represent the average percentage identified in the blood of each cohort. b) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by monocytes/LAP+ cells in a healthy control and a multiple myeloma patient. LAP: Latency-associated peptide.
Figure Legend Snippet: Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls for the expression of latency-associated peptide (LAP) on monocytes. a) Coexpression of CD14+/ LAP+. Results represent the average percentage identified in the blood of each cohort. b) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by monocytes/LAP+ cells in a healthy control and a multiple myeloma patient. LAP: Latency-associated peptide.

Techniques Used: Flow Cytometry, Cytometry, Expressing, Fluorescence

Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls. a) Coexpression of CD14+/HLA-DR+dim. b) Coexpression of CD14+/CD124+, both representing the average of myeloid-derived suppressor cell (MDSC) percentage identified in the peripheral blood of each cohort. c) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by MDSCs in monoclonal gammopathy of unknown significance, multiple myeloma, and plasma cell leukemia patients. MM: Multiple myeloma, MGUS: monoclonal gammopathy of unknown significance, MDSC: Myeloid-derived suppressor cell, LAP: latency-associated peptide, WM: Waldenström’s macroglobulinemia.
Figure Legend Snippet: Flow-cytometry analysis of peripheral blood from patients with different plasma cell dyscrasias in comparison to healthy controls. a) Coexpression of CD14+/HLA-DR+dim. b) Coexpression of CD14+/CD124+, both representing the average of myeloid-derived suppressor cell (MDSC) percentage identified in the peripheral blood of each cohort. c) An example of fluorescence activated cell scanning analysis presenting peripheral blood infiltrated by MDSCs in monoclonal gammopathy of unknown significance, multiple myeloma, and plasma cell leukemia patients. MM: Multiple myeloma, MGUS: monoclonal gammopathy of unknown significance, MDSC: Myeloid-derived suppressor cell, LAP: latency-associated peptide, WM: Waldenström’s macroglobulinemia.

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

15) Product Images from "Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health"

Article Title: Novel self-epitopes derived from aggrecan, fibrillin, and matrix metalloproteinase-3 drive distinct autoreactive T-cell responses in juvenile idiopathic arthritis and in health

Journal: Arthritis Research & Therapy

doi: 10.1186/ar2088

Cytokine production in short-term peptide-specific T-cell lines from patients with juvenile idiopathic arthritis (JIA). Short-term peptide-specific T-cell lines were generated from peripheral blood mononuclear cells of five patients with polyarticular JIA. After 14 days of culture, supernatants were taken for multiplex cytokine analysis and cells were stained for fluorescence-activated cell sorting (FACS) analysis. The aggrecan peptide induced significant production of interferon-γ (IFN-γ)/interleukin (IL)-17 and inhibition of IL-10 production ( p
Figure Legend Snippet: Cytokine production in short-term peptide-specific T-cell lines from patients with juvenile idiopathic arthritis (JIA). Short-term peptide-specific T-cell lines were generated from peripheral blood mononuclear cells of five patients with polyarticular JIA. After 14 days of culture, supernatants were taken for multiplex cytokine analysis and cells were stained for fluorescence-activated cell sorting (FACS) analysis. The aggrecan peptide induced significant production of interferon-γ (IFN-γ)/interleukin (IL)-17 and inhibition of IL-10 production ( p

Techniques Used: Generated, Multiplex Assay, Staining, Fluorescence, FACS, Inhibition

16) Product Images from "Reduced thymic output, cell cycle abnormalities, and increased apoptosis of T lymphocytes in patients with cartilage-hair hypoplasia"

Article Title: Reduced thymic output, cell cycle abnormalities, and increased apoptosis of T lymphocytes in patients with cartilage-hair hypoplasia

Journal: The Journal of allergy and clinical immunology

doi: 10.1016/j.jaci.2011.03.042

Analysis of apoptosis and cell death. Left , Representative example of fluorescence-activated cell sorting plots after staining of PBMCs for AnnV and 7-AAD in healthy controls and patients with CHH on in vitro stimulation with anti-CD3 ( top ) or anti-CD3
Figure Legend Snippet: Analysis of apoptosis and cell death. Left , Representative example of fluorescence-activated cell sorting plots after staining of PBMCs for AnnV and 7-AAD in healthy controls and patients with CHH on in vitro stimulation with anti-CD3 ( top ) or anti-CD3

Techniques Used: Fluorescence, FACS, Staining, In Vitro

17) Product Images from "Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes"

Article Title: Engineering human ventricular heart muscles based on a highly efficient system for purification of human pluripotent stem cell-derived ventricular cardiomyocytes

Journal: Stem Cell Research & Therapy

doi: 10.1186/s13287-017-0651-x

Effective enrichment of MLC-2v-positive human early ventricular cardiomyocytes based on the EGFP selection system. a FACS sorting showing positive cardiomyocytes derived from MYL2 EGFP/w hESCs 25 days after cardiac differentiation. b Representative green fluorescence (EGFP), bright field (BFEGFP), and merged images of MYL2 EGFP/w hESC-derived cardiomyocytes before and after FACS sorting. Scale bars, 100 μm. c Immunofluorescence microscopy showing coexpression of EGFP and MLC-2v in FACS sorted MYL2 EGFP/w hESC-derived cardiomyocytes. Scale bars, 50 μm. d Double immunofluorescence staining showing coexpression of EGFP and MLC-2v in FACS sorted MYL2 EGFP/w hESC-derived cardiomyocytes (1). MLC-2v was expressed in all of the cTnT-positive MYL2 EGFP/w hESC-derived cardiomyocytes post FACS (2). MLC-2a only showed background expression level in the cTnT-positive MYL2 EGFP/w hESC-derived cardiomyocytes post FACS (3). Scale bars, 100 μm. e Vitality assessment of the FACS selected MYL2 EGFP/w -CMs by live cell staining for markers of apoptosis. Representative graph as detected by flow cytometry analysis. f Percentages of ventricular-like, atrial-like, and nodal-like cells produced from MYL2 EGFP/w -CMs before and after FACS sorting as determined by single cell patch clamp. EGFP enhanced green fluorescent protein, MLC-2v cardiac ventricular isoform of myosin light chain-2, V-like ventricular-like cells, A-like atrial-like cells, N-like nodal-like cells
Figure Legend Snippet: Effective enrichment of MLC-2v-positive human early ventricular cardiomyocytes based on the EGFP selection system. a FACS sorting showing positive cardiomyocytes derived from MYL2 EGFP/w hESCs 25 days after cardiac differentiation. b Representative green fluorescence (EGFP), bright field (BFEGFP), and merged images of MYL2 EGFP/w hESC-derived cardiomyocytes before and after FACS sorting. Scale bars, 100 μm. c Immunofluorescence microscopy showing coexpression of EGFP and MLC-2v in FACS sorted MYL2 EGFP/w hESC-derived cardiomyocytes. Scale bars, 50 μm. d Double immunofluorescence staining showing coexpression of EGFP and MLC-2v in FACS sorted MYL2 EGFP/w hESC-derived cardiomyocytes (1). MLC-2v was expressed in all of the cTnT-positive MYL2 EGFP/w hESC-derived cardiomyocytes post FACS (2). MLC-2a only showed background expression level in the cTnT-positive MYL2 EGFP/w hESC-derived cardiomyocytes post FACS (3). Scale bars, 100 μm. e Vitality assessment of the FACS selected MYL2 EGFP/w -CMs by live cell staining for markers of apoptosis. Representative graph as detected by flow cytometry analysis. f Percentages of ventricular-like, atrial-like, and nodal-like cells produced from MYL2 EGFP/w -CMs before and after FACS sorting as determined by single cell patch clamp. EGFP enhanced green fluorescent protein, MLC-2v cardiac ventricular isoform of myosin light chain-2, V-like ventricular-like cells, A-like atrial-like cells, N-like nodal-like cells

Techniques Used: Selection, FACS, Derivative Assay, Fluorescence, Immunofluorescence, Microscopy, Double Immunofluorescence Staining, Expressing, Staining, Flow Cytometry, Cytometry, Produced, Patch Clamp

18) Product Images from "Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells"

Article Title: Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells

Journal: Cellular and Molecular Immunology

doi: 10.1038/cmi.2016.63

Attenuated inflammatory cell infiltration in ST2-KO mice in bile-duct ligation (BDL)-induced acute inflammation. Infiltrating mononuclear cells were isolated from the livers of WT or ST2-KO mice 1 day after BDL. Cells were stained by specific antibodies and analyzed by fluorescence activated cell sorting. ( a ) Proportion of Th1 (CD4 + IFNγ + ) cells, Th2 (CD4 + IL-4 + ) cells, neutrophils and myeloid cells (Gr-1 hi CD11b + ) and macrophages (CD11b + F4/80) were upregulated after BDL but significantly reduced in ST2-deficient mice. ( b ) ILC2s (group 2 innate lymphoid cells), identified as lineage - Sca-1 + and c-kit 1 + , were significantly increased 21 days after BDL. The data represent percentages and are from one representative experiment of three independent studies.
Figure Legend Snippet: Attenuated inflammatory cell infiltration in ST2-KO mice in bile-duct ligation (BDL)-induced acute inflammation. Infiltrating mononuclear cells were isolated from the livers of WT or ST2-KO mice 1 day after BDL. Cells were stained by specific antibodies and analyzed by fluorescence activated cell sorting. ( a ) Proportion of Th1 (CD4 + IFNγ + ) cells, Th2 (CD4 + IL-4 + ) cells, neutrophils and myeloid cells (Gr-1 hi CD11b + ) and macrophages (CD11b + F4/80) were upregulated after BDL but significantly reduced in ST2-deficient mice. ( b ) ILC2s (group 2 innate lymphoid cells), identified as lineage - Sca-1 + and c-kit 1 + , were significantly increased 21 days after BDL. The data represent percentages and are from one representative experiment of three independent studies.

Techniques Used: Mouse Assay, Ligation, Isolation, Staining, Fluorescence, FACS

19) Product Images from "AST-120 Reduces Neuroinflammation Induced by Indoxyl Sulfate in Glial Cells"

Article Title: AST-120 Reduces Neuroinflammation Induced by Indoxyl Sulfate in Glial Cells

Journal: Journal of Clinical Medicine

doi: 10.3390/jcm7100365

Effect of IS (60, 30 and 15 μM) in inflammatory conditions, induced by LPS (1 µg/mL) + IFN (100 U/mL), on NO relase ( Panel A ), evaluated as NO 2 [µM]. Effect of IS (15–60 μM) in presence of LPS (1 µg/mL) + IFN (100 U/mL) on iNOS ( Panel B ), and COX-2 ( Panel D ) expression in primary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson), and elaborated with Cell Quest software. Panel C shows the representative fluorescence images for iNOS expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM + LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the representative fluorescence images for COX-2 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM with LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM with LPS + IFN. Effect of IS (15, 30, and 60 μM) in inflammatory conditions, induced by LPS (1 µg/mL) and IFN (100 U/mL) on TNF-α ( Panel F ) and on IL-6 ( Panel G ) release by astrocytes and mixed glial cells. Cyokine release was assessed by ELISA assay. Values are expressed as NO 2 - release, or mean fluorescence intensity or as pg/mL protein for cytokines. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°° denotes p
Figure Legend Snippet: Effect of IS (60, 30 and 15 μM) in inflammatory conditions, induced by LPS (1 µg/mL) + IFN (100 U/mL), on NO relase ( Panel A ), evaluated as NO 2 [µM]. Effect of IS (15–60 μM) in presence of LPS (1 µg/mL) + IFN (100 U/mL) on iNOS ( Panel B ), and COX-2 ( Panel D ) expression in primary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson), and elaborated with Cell Quest software. Panel C shows the representative fluorescence images for iNOS expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM + LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the representative fluorescence images for COX-2 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM with LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM with LPS + IFN. Effect of IS (15, 30, and 60 μM) in inflammatory conditions, induced by LPS (1 µg/mL) and IFN (100 U/mL) on TNF-α ( Panel F ) and on IL-6 ( Panel G ) release by astrocytes and mixed glial cells. Cyokine release was assessed by ELISA assay. Values are expressed as NO 2 - release, or mean fluorescence intensity or as pg/mL protein for cytokines. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°° denotes p

Techniques Used: Expressing, Fluorescence, FACS, Software, Enzyme-linked Immunosorbent Assay

Effect of IS (60, 30 and 15 μM) on primary CNS cells treated with LPS (1 µg/mL) + IFN (100 U/mL) on nitrotyrosine formation ( Panel A ), and on ROS release ( Panel B ), evaluated by means of the probe 2’,7’ dichlorofluorescein-diacetate (H 2 DCF-DA) and on HO-1 expression ( Panel C ) in prmary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Panel D shows the representative fluorescence images for HO-1 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM in presence of LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the effect of supernatant from IS-treated microglia on cortical and hippocampal neuronal cell viability. Values are expressed as mean fluorescence intensity or as cell citotoxicity. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°°, °° denote p
Figure Legend Snippet: Effect of IS (60, 30 and 15 μM) on primary CNS cells treated with LPS (1 µg/mL) + IFN (100 U/mL) on nitrotyrosine formation ( Panel A ), and on ROS release ( Panel B ), evaluated by means of the probe 2’,7’ dichlorofluorescein-diacetate (H 2 DCF-DA) and on HO-1 expression ( Panel C ) in prmary astrocytes and mixed glial cells. Cellular fluorescence was evaluated using fluorescence-activated cell sorting analysis (FACSscan; Becton Dickinson) and elaborated with Cell Quest software. Panel D shows the representative fluorescence images for HO-1 expression (for primary astrocytes the pink line represent the cellular control, the blue line represent LPS + IFN, the yellow line represent IS 30 µM in presence of LPS + IFN; for mixed glial cells the violet line represent the cellular control, the light blue line represent LPS + IFN, the orange line represent IS 30 µM + LPS + IFN). Panel E shows the effect of supernatant from IS-treated microglia on cortical and hippocampal neuronal cell viability. Values are expressed as mean fluorescence intensity or as cell citotoxicity. Comparisons were performed using a one-way analysis of variance and multiple comparisons were made by Bonferroni’s post test. °°°, °° denote p

Techniques Used: Expressing, Fluorescence, FACS, Software

20) Product Images from "Elevation of adenylate energy charge by angiopoietin-like 4 enhances epithelial–mesenchymal transition by inducing 14-3-3γ expression"

Article Title: Elevation of adenylate energy charge by angiopoietin-like 4 enhances epithelial–mesenchymal transition by inducing 14-3-3γ expression

Journal: Oncogene

doi: 10.1038/onc.2017.244

ANGPTL4 increases cellular bioenergetics for EMT competency. ( a ) Fluorescence-activated cell sorting (FACS) analysis of the fluorescent glucose analog 2-NBDG uptake and ( b ) percentage increase in energy charge in MKN74 and MKN74 Snai1ER cells after the indicated treatments. ( c ) Immunoblot analysis of GLUT1 expression in MKN74 cells after the indicated treatments. ( d , f ) Immunofluorescence staining of E-cadherin in hypoxia-treated MKN74 cells in the presence of neutralizing human cANGPTL4 antibodies (α-cANGPTL4) ( d ) and recombinant human cANGPTL4 (rh-cANGPTL4) -treated MKN74 ( f ) at the indicated time intervals. Cells were counterstained with DAPI (blue) for nuclei and phalloidin (red) for actin cytoskeleton. Scale bar=40 μm. ( e , g ) Relative mRNA expression (left panel) and immunodetection (middle and right panels) of EMT markers in hypoxia-treated MKN74 cells exposed to α-cANGPTL4 ( e ) and rh-cANGPTL4 ( g ) at the indicated time intervals. For immunoblot analyses, representative immunoblot pictures and densitometric quantification plots are shown. Loading controls for the immunoblot analyses were from the same sample. For qPCR, TBP was used as reference gene. Data are represented as mean±s.d. from at least three independent experiments. * P
Figure Legend Snippet: ANGPTL4 increases cellular bioenergetics for EMT competency. ( a ) Fluorescence-activated cell sorting (FACS) analysis of the fluorescent glucose analog 2-NBDG uptake and ( b ) percentage increase in energy charge in MKN74 and MKN74 Snai1ER cells after the indicated treatments. ( c ) Immunoblot analysis of GLUT1 expression in MKN74 cells after the indicated treatments. ( d , f ) Immunofluorescence staining of E-cadherin in hypoxia-treated MKN74 cells in the presence of neutralizing human cANGPTL4 antibodies (α-cANGPTL4) ( d ) and recombinant human cANGPTL4 (rh-cANGPTL4) -treated MKN74 ( f ) at the indicated time intervals. Cells were counterstained with DAPI (blue) for nuclei and phalloidin (red) for actin cytoskeleton. Scale bar=40 μm. ( e , g ) Relative mRNA expression (left panel) and immunodetection (middle and right panels) of EMT markers in hypoxia-treated MKN74 cells exposed to α-cANGPTL4 ( e ) and rh-cANGPTL4 ( g ) at the indicated time intervals. For immunoblot analyses, representative immunoblot pictures and densitometric quantification plots are shown. Loading controls for the immunoblot analyses were from the same sample. For qPCR, TBP was used as reference gene. Data are represented as mean±s.d. from at least three independent experiments. * P

Techniques Used: Fluorescence, FACS, Expressing, Immunofluorescence, Staining, Recombinant, Immunodetection, Real-time Polymerase Chain Reaction

Increased cellular metabolic activity is essential for epithelial–mesenchymal transition. ( a ) Percentage increase in energy charge in breast, colorectal, gastric and head and neck tissues at various tumor stages. Comparison was made against respective stage I samples. ( b ) Immunoblot analysis of cANGPTL4 expression in patient biopsies at various tumor stages. ( c ) Immunofluorescence staining of E-cadherin (green) in hypoxia cells at the indicated time intervals. Cells were counterstained with DAPI (blue) for nuclei and phalloidin (red) for actin cytoskeleton. Scale bar=40 μm. ( d ) Relative mRNA expression of EMT markers (CDH1, DDR1, ERBB3, Snai1 and ZEB1) in hypoxia-exposed MKN74 cells at indicated time points. ( e ) Immunoblot analysis of E-cadherin and Snai2 expression in hypoxia-exposed MKN74 cells at indicated time points. ( f ) Fluorescence-activated cell sorting (FACS) analysis of the fluorescent glucose analog 2-NBDG uptake and ( g ) percentage increase in energy charge in MKN74 and MKN74 Snai1ER cells after the indicated treatments. ( h ) Relative mRNA expression (left panel) and immunoblot analysis (right panel) of ANGPTL4 in MKN74 and MKN74 Snai1ER cells after the indicated treatments. ( i ) Percentage increase in energy change in MCF7, HSC, II4 and HepG2 cells after indicated treatments. For immunoblot analyses, representative immunoblot pictures and densitometric quantification plots are shown. Loading controls for the immunoblot analyses were from the same sample. For qPCR, TBP was used as reference gene. Data are represented as mean±s.d. from at least three independent experiments. * P
Figure Legend Snippet: Increased cellular metabolic activity is essential for epithelial–mesenchymal transition. ( a ) Percentage increase in energy charge in breast, colorectal, gastric and head and neck tissues at various tumor stages. Comparison was made against respective stage I samples. ( b ) Immunoblot analysis of cANGPTL4 expression in patient biopsies at various tumor stages. ( c ) Immunofluorescence staining of E-cadherin (green) in hypoxia cells at the indicated time intervals. Cells were counterstained with DAPI (blue) for nuclei and phalloidin (red) for actin cytoskeleton. Scale bar=40 μm. ( d ) Relative mRNA expression of EMT markers (CDH1, DDR1, ERBB3, Snai1 and ZEB1) in hypoxia-exposed MKN74 cells at indicated time points. ( e ) Immunoblot analysis of E-cadherin and Snai2 expression in hypoxia-exposed MKN74 cells at indicated time points. ( f ) Fluorescence-activated cell sorting (FACS) analysis of the fluorescent glucose analog 2-NBDG uptake and ( g ) percentage increase in energy charge in MKN74 and MKN74 Snai1ER cells after the indicated treatments. ( h ) Relative mRNA expression (left panel) and immunoblot analysis (right panel) of ANGPTL4 in MKN74 and MKN74 Snai1ER cells after the indicated treatments. ( i ) Percentage increase in energy change in MCF7, HSC, II4 and HepG2 cells after indicated treatments. For immunoblot analyses, representative immunoblot pictures and densitometric quantification plots are shown. Loading controls for the immunoblot analyses were from the same sample. For qPCR, TBP was used as reference gene. Data are represented as mean±s.d. from at least three independent experiments. * P

Techniques Used: Activity Assay, Expressing, Immunofluorescence, Staining, Fluorescence, FACS, Real-time Polymerase Chain Reaction

21) Product Images from "The impact of TEL-AML1 (ETV6-RUNX1) expression in precursor B cells and implications for leukaemia using three different genome-wide screening methods"

Article Title: The impact of TEL-AML1 (ETV6-RUNX1) expression in precursor B cells and implications for leukaemia using three different genome-wide screening methods

Journal: Blood Cancer Journal

doi: 10.1038/bcj.2013.48

TEL-AML1 antibody design and specificity testing. ( a ) Design strategy for the TEL-AML1 antibody. The immunization peptide spanning the fusion site between the TEL (white) and AML1 (black) fusion partners is indicated. ( b ) Specificity of the TEL-AML1 antibody. Western blots (WB) of the parental BA/F3 cell line (TA−) and stable cell lines carrying the inducible TEL-AML1 fusion construct (TA+) are treated with mifepristone as indicated. TEL-AML1 was specifically detected only in the induced cell lines, whereas the AML and TEL antibodies (right panels) detected both, the fusion protein and the native protein. Please note that the TEL antibody also detects numerous unspecific bands in the whole-cell lysates. ( c ) Detection of TEL-AML1 fusion protein by fluorescence-activated cell sorting (FACS) analysis. Induction with mifepriston resulted in on average 93.5±0.7% ( n =15;±1 s.d.) cells carrying the TEL-AML1 fusion protein in FACS analysis using the TEL-AML1 antibody. A representative example is shown. Tightness of the induction system is shown in comparison to parental BA/F3 cells treated with mifepriston. Abbreviations: FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
Figure Legend Snippet: TEL-AML1 antibody design and specificity testing. ( a ) Design strategy for the TEL-AML1 antibody. The immunization peptide spanning the fusion site between the TEL (white) and AML1 (black) fusion partners is indicated. ( b ) Specificity of the TEL-AML1 antibody. Western blots (WB) of the parental BA/F3 cell line (TA−) and stable cell lines carrying the inducible TEL-AML1 fusion construct (TA+) are treated with mifepristone as indicated. TEL-AML1 was specifically detected only in the induced cell lines, whereas the AML and TEL antibodies (right panels) detected both, the fusion protein and the native protein. Please note that the TEL antibody also detects numerous unspecific bands in the whole-cell lysates. ( c ) Detection of TEL-AML1 fusion protein by fluorescence-activated cell sorting (FACS) analysis. Induction with mifepriston resulted in on average 93.5±0.7% ( n =15;±1 s.d.) cells carrying the TEL-AML1 fusion protein in FACS analysis using the TEL-AML1 antibody. A representative example is shown. Tightness of the induction system is shown in comparison to parental BA/F3 cells treated with mifepriston. Abbreviations: FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Techniques Used: Western Blot, Stable Transfection, Construct, Fluorescence, FACS

22) Product Images from "The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors"

Article Title: The expression and significance of insulin-like growth factor-1 receptor and its pathway on breast cancer stem/progenitors

Journal: Breast Cancer Research : BCR

doi: 10.1186/bcr3423

Insulin-like growth factor 1 receptor serves as a marker for breast cancer stem cells . (A) Cells from xenograft BC0145 or BC0244 tumors were sorted as indicated populations and phosphorylation of insulin-like growth factor 1 receptor (IGF-1R) was determined by western blot. (B) pIGF-1R Tyr1165/1166 of immunoprecipitated IGF-1R from aldehyde dehydrogenase (ALDH) - or ALDH + BC0244 xenograft tumor cells was determined. (C) Tumor cells of BC0145 or BC0244 xenografts were stained with PE-conjugated anti-IGF-1R antibody and FITC-conjugated anti-H2K d antibody. CD24 - CD44 + or ALDH + cells within IGF-1R + /IGF-1R - BC0145 cells (upper panel in (B)) or IGF-1R hi /IGF-1R lo BC0244 cells (lower panel in (B)) were determined by co-stain with PE-Cy7-conjugated anti-CD24/APC-conjugated anti-CD44 antibodies or Aldefluor substrate. (D), (E) Two populations of IGF-1R + /IGF-1R - (BC0145) or IGF-1R hi /IGF-1R lo (BC0244) cells were sorted from the H2K d- population by fluorescence-activated cell sorting (FACS) and determined the mammosphere formation capability (D) or tumorigenicity (E). The CSC frequency was calculated by ELDA software (table in (E)). All experiments were repeated independently at least twice and results shown were from a representative experiment. IP, immunoprecipitation; IB, immunoblot.
Figure Legend Snippet: Insulin-like growth factor 1 receptor serves as a marker for breast cancer stem cells . (A) Cells from xenograft BC0145 or BC0244 tumors were sorted as indicated populations and phosphorylation of insulin-like growth factor 1 receptor (IGF-1R) was determined by western blot. (B) pIGF-1R Tyr1165/1166 of immunoprecipitated IGF-1R from aldehyde dehydrogenase (ALDH) - or ALDH + BC0244 xenograft tumor cells was determined. (C) Tumor cells of BC0145 or BC0244 xenografts were stained with PE-conjugated anti-IGF-1R antibody and FITC-conjugated anti-H2K d antibody. CD24 - CD44 + or ALDH + cells within IGF-1R + /IGF-1R - BC0145 cells (upper panel in (B)) or IGF-1R hi /IGF-1R lo BC0244 cells (lower panel in (B)) were determined by co-stain with PE-Cy7-conjugated anti-CD24/APC-conjugated anti-CD44 antibodies or Aldefluor substrate. (D), (E) Two populations of IGF-1R + /IGF-1R - (BC0145) or IGF-1R hi /IGF-1R lo (BC0244) cells were sorted from the H2K d- population by fluorescence-activated cell sorting (FACS) and determined the mammosphere formation capability (D) or tumorigenicity (E). The CSC frequency was calculated by ELDA software (table in (E)). All experiments were repeated independently at least twice and results shown were from a representative experiment. IP, immunoprecipitation; IB, immunoblot.

Techniques Used: Marker, Western Blot, Immunoprecipitation, Staining, Fluorescence, FACS, Software

23) Product Images from "Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo"

Article Title: Histone deacetylase inhibitor thailandepsin-A activates Notch signaling and suppresses neuroendocrine cancer cell growth in vivo

Journal: Oncotarget

doi: 10.18632/oncotarget.19993

Cell cycle arrest in NE cancer cells caused by TDP-A treatment (A) Detection of p21, p27, cyclin B1, and cyclin D1 protein expression by Western blot in NE cancer cells treated with multiple concentrations of TDP-A (0-8nM) or vehicle control (DMSO). Equal loading was confirmed with GAPDH. (B) NE cancer cells were treated with two doses of TDP-A and vehicle control (DMSO) for 48 hrs, and stained with propidium iodide (3 ug/mL). Cell cycle analysis with fluorescence-activated cell sorting showed that the mechanism of growth inhibition is by cell cycle arrest at G2/M phase in BON cells and at G1 phase in MZ and TT cells.
Figure Legend Snippet: Cell cycle arrest in NE cancer cells caused by TDP-A treatment (A) Detection of p21, p27, cyclin B1, and cyclin D1 protein expression by Western blot in NE cancer cells treated with multiple concentrations of TDP-A (0-8nM) or vehicle control (DMSO). Equal loading was confirmed with GAPDH. (B) NE cancer cells were treated with two doses of TDP-A and vehicle control (DMSO) for 48 hrs, and stained with propidium iodide (3 ug/mL). Cell cycle analysis with fluorescence-activated cell sorting showed that the mechanism of growth inhibition is by cell cycle arrest at G2/M phase in BON cells and at G1 phase in MZ and TT cells.

Techniques Used: Expressing, Western Blot, Staining, Cell Cycle Assay, Fluorescence, FACS, Inhibition

24) Product Images from "The natural organosulfur compound dipropyltetrasulfide prevents HOCl-induced systemic sclerosis in the mouse"

Article Title: The natural organosulfur compound dipropyltetrasulfide prevents HOCl-induced systemic sclerosis in the mouse

Journal: Arthritis Research & Therapy

doi: 10.1186/ar4351

Proapoptotic effects of DPTTS analyzed with fluorescence-activated cells sorting. (a) Normal and HOCl fibroblasts were incubated with 40 μ M DPTTS for 5, 10, or 15 hours. The ratio of apoptosis to necrosis was analyzed with flow cytometry by using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO-1 and propidium iodide. Necrotic cells were PI positive, and apoptotic cells were YO-PRO-1 positive. One of three representative experiments is shown. (b) Kinetics of the apoptosis/necrosis ratio of normal and HOCl fibroblasts treated with 40 μ M DPTTS.
Figure Legend Snippet: Proapoptotic effects of DPTTS analyzed with fluorescence-activated cells sorting. (a) Normal and HOCl fibroblasts were incubated with 40 μ M DPTTS for 5, 10, or 15 hours. The ratio of apoptosis to necrosis was analyzed with flow cytometry by using the Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO-1 and propidium iodide. Necrotic cells were PI positive, and apoptotic cells were YO-PRO-1 positive. One of three representative experiments is shown. (b) Kinetics of the apoptosis/necrosis ratio of normal and HOCl fibroblasts treated with 40 μ M DPTTS.

Techniques Used: Fluorescence, Incubation, Flow Cytometry, Cytometry, Permeability

25) Product Images from "Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of ?2?1 Integrin Complex *"

Article Title: Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of ?2?1 Integrin Complex *

Journal:

doi: 10.1074/jbc.M109.010934

Establishment of PC3 and LNCaP cells expressing core3 synthase. A , PC3 and LNCaP cells selected after transfection of core3 synthase and Geneticin resistance markers were subjected to fluorescence-activated cell sorting analysis after neuraminidase treatment
Figure Legend Snippet: Establishment of PC3 and LNCaP cells expressing core3 synthase. A , PC3 and LNCaP cells selected after transfection of core3 synthase and Geneticin resistance markers were subjected to fluorescence-activated cell sorting analysis after neuraminidase treatment

Techniques Used: Expressing, Transfection, Fluorescence, FACS

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Flow Cytometry:

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Cytometry:

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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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Construct:

Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct
Article Snippet: .. Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) . ..

Incubation:

Article Title: Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells
Article Snippet: .. The trypsinized individual cells were incubated with the FITC-conjugated and PE-conjugated monoclonal antibodies: antihuman CD29, antihuman CD105, antihuman CD44, antihuman CD90, antihuman CD34, and antihuman CD45 (BD Biosciences, San Jose, CA) at 4°C for 30 min. Then the cells were washed three times with PBS and analyzed by flow cytometry analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA). .. Hematopoietic colony assays were performed in 35-mm low-adhesion plastic dishes using MethoCult GF-H4434 semisolid medium (Stem Cell Technologies, Vancouver, Canada) consisting of 1% methylcellulose, 30% FBS, 1% bovine serum albumin (BSA), 50 ng/mL stem cell factor, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 20 ng/mL granulocyte colony-stimulating factor (G-CSF), 20 ng/mL interleukin-3 (IL-3), 20 ng/mL interleukin-6 (IL-6), and 3 U/mL EPO.

Expressing:

Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct
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Staining:

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis
Article Snippet: .. Then, the cells were stained with PerCP-labeled anti-mouse CD4 (eBioscience) and FITC-conjugated anti-mouse CD25 (eBioscience) at 4 °C for 40 min. After washing, fixing and permeabilization, the cells were stained intracellularly with PE-conjugated anti-mouse Foxp3 (eBioscience) at 4 °C for 1 hour before by flow cytometry analysis on a FACS Calibur machine (BD Biosciences). .. Data were analyzed with CellQuest software (BD Biosciences).

FACS:

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis
Article Snippet: .. Then, the cells were stained with PerCP-labeled anti-mouse CD4 (eBioscience) and FITC-conjugated anti-mouse CD25 (eBioscience) at 4 °C for 40 min. After washing, fixing and permeabilization, the cells were stained intracellularly with PE-conjugated anti-mouse Foxp3 (eBioscience) at 4 °C for 1 hour before by flow cytometry analysis on a FACS Calibur machine (BD Biosciences). .. Data were analyzed with CellQuest software (BD Biosciences).

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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    Becton Dickinson mapk pathway activation analysis
    Percentage of p-ERK, p-JNK, p-p38 <t>MAPK</t> positive keratinocytes. Results are presented as the mean ± SD [%]. Data acquired by flow <t>cytometry.</t> n = 18. Results were considered significant for p
    Mapk Pathway Activation Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mapk pathway activation analysis/product/Becton Dickinson
    Average 92 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    mapk pathway activation analysis - by Bioz Stars, 2020-11
    92/100 stars
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    95
    Becton Dickinson flow cytometry analysis
    Expression of SAF-1 and SAF-3 variants driven by alternative promoters The biochromatic fluorescent reporters were driven by −1692/+277 promoter of SAF/MAZ gene or −1401/−277, −595/+277, and −1692/+277Δ−1401/−595 SAF-1. The average FIs of cells were analyzed for SAF-1 and SAF-3 by flow <t>cytometry</t> ( A ).The data shown represent the difference of mean ±SEM of three separate expreiments between two groups indicated by line below star symbols (** P
    Flow Cytometry Analysis, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 95/100, based on 2610 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/flow cytometry analysis/product/Becton Dickinson
    Average 95 stars, based on 2610 article reviews
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    flow cytometry analysis - by Bioz Stars, 2020-11
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    Image Search Results


    Percentage of p-ERK, p-JNK, p-p38 MAPK positive keratinocytes. Results are presented as the mean ± SD [%]. Data acquired by flow cytometry. n = 18. Results were considered significant for p

    Journal: PLoS ONE

    Article Title: Fas/FasL pathway and cytokines in keratinocytes in atopic dermatitis – Manipulation by the electromagnetic field

    doi: 10.1371/journal.pone.0205103

    Figure Lengend Snippet: Percentage of p-ERK, p-JNK, p-p38 MAPK positive keratinocytes. Results are presented as the mean ± SD [%]. Data acquired by flow cytometry. n = 18. Results were considered significant for p

    Article Snippet: MAPK pathway activation analysis was performed using flow cytometry (FACS Calibur, BD, USA) and phospho-specific antibodies (Phospho-ERK1/2 (Thr202, Tyr204) Monoclonal Antibody (MILAN8R), Phospho-p38 MAPK (Thr180, Tyr182) Monoclonal Antibody (4NIT4KK) and Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Monoclonal Antibody).

    Techniques: Flow Cytometry, Cytometry

    Expression of SAF-1 and SAF-3 variants driven by alternative promoters The biochromatic fluorescent reporters were driven by −1692/+277 promoter of SAF/MAZ gene or −1401/−277, −595/+277, and −1692/+277Δ−1401/−595 SAF-1. The average FIs of cells were analyzed for SAF-1 and SAF-3 by flow cytometry ( A ).The data shown represent the difference of mean ±SEM of three separate expreiments between two groups indicated by line below star symbols (** P

    Journal: Bioscience Reports

    Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct

    doi: 10.1042/BSR20171668

    Figure Lengend Snippet: Expression of SAF-1 and SAF-3 variants driven by alternative promoters The biochromatic fluorescent reporters were driven by −1692/+277 promoter of SAF/MAZ gene or −1401/−277, −595/+277, and −1692/+277Δ−1401/−595 SAF-1. The average FIs of cells were analyzed for SAF-1 and SAF-3 by flow cytometry ( A ).The data shown represent the difference of mean ±SEM of three separate expreiments between two groups indicated by line below star symbols (** P

    Article Snippet: Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) .

    Techniques: Expressing, Flow Cytometry, Cytometry

    Repression of SAF-1 and SAF-3 promoter by transcription factor Elk-1 and endogenous SAF-1/SAF-3 expression Elk-1 cis -element on SAF/MAZ promoter was identified by EMSA ( A ). Horizontal black and red arrows represent the specific protein/DNA binding bands and anti-His/His tagged ELK-1/DNA probe supershift bands, respectively. The bichromatic fluorescent reporter plasmids were transiently co-transfected with empty plasmid, pCGN-Elk-1 and pN3-Sp1 into HeLa cells. The SAF-1 and SAF-3 promoter activation status were tested by either laser co-focus microscopy ( B ) or by flow cytometry analysis ( C ). The endogenous SAF-1 and SAF-3 mRNA expression status in HeLa cells are shown by ( D ). In (D), lanes 1 and 8 are DNA markers, lanes 2 and 5 are SAF-1 mRNA levels (271 bp), lanes 3 and 6 are SAF-3 mRNA levels (208 bp), and lanes 4 and 7 are GAPDH mRNA levels (177 bp). Abbreviation: EMSA, electrophoretic mobility shift assay.

    Journal: Bioscience Reports

    Article Title: Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct

    doi: 10.1042/BSR20171668

    Figure Lengend Snippet: Repression of SAF-1 and SAF-3 promoter by transcription factor Elk-1 and endogenous SAF-1/SAF-3 expression Elk-1 cis -element on SAF/MAZ promoter was identified by EMSA ( A ). Horizontal black and red arrows represent the specific protein/DNA binding bands and anti-His/His tagged ELK-1/DNA probe supershift bands, respectively. The bichromatic fluorescent reporter plasmids were transiently co-transfected with empty plasmid, pCGN-Elk-1 and pN3-Sp1 into HeLa cells. The SAF-1 and SAF-3 promoter activation status were tested by either laser co-focus microscopy ( B ) or by flow cytometry analysis ( C ). The endogenous SAF-1 and SAF-3 mRNA expression status in HeLa cells are shown by ( D ). In (D), lanes 1 and 8 are DNA markers, lanes 2 and 5 are SAF-1 mRNA levels (271 bp), lanes 3 and 6 are SAF-3 mRNA levels (208 bp), and lanes 4 and 7 are GAPDH mRNA levels (177 bp). Abbreviation: EMSA, electrophoretic mobility shift assay.

    Article Snippet: Although there were statistically meaningful differences amongst these four constructs for SAF-1 expression by flow cytometry analysis, the change of SAF-1 expression was not as obvious as for SAF-3 expression ( A) .

    Techniques: Expressing, Binding Assay, Transfection, Plasmid Preparation, Activation Assay, Microscopy, Flow Cytometry, Cytometry, Electrophoretic Mobility Shift Assay

    Actinomycin D induces apoptosis of endothelial cells. (a) BAECs were exposed to actinomycin D (1 μ g ml −1 ) for the time indicated, and cell death was assessed by PI staining using flow cytometry. Values showing the apoptosis induced by actinomycin D (apoptosis in the presence of actinomycin D minus basal apoptosis) are mean±s.e.m. ( n =30). (b) Morphological changes observed by light microscopy of actinomycin D-treated cells (right) versus control cells (left). Arrows on the right-hand panel point to cell shrinkage (1) and membrane blebbing (2), characteristic features of apoptotic cell death.

    Journal: British Journal of Pharmacology

    Article Title: Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis

    doi: 10.1038/sj.bjp.0705347

    Figure Lengend Snippet: Actinomycin D induces apoptosis of endothelial cells. (a) BAECs were exposed to actinomycin D (1 μ g ml −1 ) for the time indicated, and cell death was assessed by PI staining using flow cytometry. Values showing the apoptosis induced by actinomycin D (apoptosis in the presence of actinomycin D minus basal apoptosis) are mean±s.e.m. ( n =30). (b) Morphological changes observed by light microscopy of actinomycin D-treated cells (right) versus control cells (left). Arrows on the right-hand panel point to cell shrinkage (1) and membrane blebbing (2), characteristic features of apoptotic cell death.

    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.).

    Techniques: Staining, Flow Cytometry, Cytometry, Light Microscopy

    Flow cytometry analysis of hematopoietic differentiation of EBs. ( A ) Generation of EBs from hESCs (scale bar=100 μm). ( B ) Flow cytometric analysis of CD34 + and CD45 + antigen at different inducing times. ( C ) Statistical analysis for the cell surface antigen expression of cells in different groups. The data represent the mean±SEM from three experiments.

    Journal: Cellular Reprogramming

    Article Title: Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells

    doi: 10.1089/cell.2011.0013

    Figure Lengend Snippet: Flow cytometry analysis of hematopoietic differentiation of EBs. ( A ) Generation of EBs from hESCs (scale bar=100 μm). ( B ) Flow cytometric analysis of CD34 + and CD45 + antigen at different inducing times. ( C ) Statistical analysis for the cell surface antigen expression of cells in different groups. The data represent the mean±SEM from three experiments.

    Article Snippet: The trypsinized individual cells were incubated with the FITC-conjugated and PE-conjugated monoclonal antibodies: antihuman CD29, antihuman CD105, antihuman CD44, antihuman CD90, antihuman CD34, and antihuman CD45 (BD Biosciences, San Jose, CA) at 4°C for 30 min. Then the cells were washed three times with PBS and analyzed by flow cytometry analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA).

    Techniques: Flow Cytometry, Cytometry, Expressing

    Morphology and identification of EPO overexpressing hFLSCs. ( A ) The of human fetal liver stromal cells (hFLSCs) show typical fibroblast morphology(scale bar=100 μm). ( B ) The growth curve of hFLSCs expressing EPO. The status of growth of cells indicated little difference between hFLSCs expressing ectopic EPO and control FLSCs. ( C ) Expression of enhanced green fluorescence protein (eGFP) in hFLSCs under the fluorescence (scale bar=100 μm). ( D ) Cell phenotype analysis of hFLSCs by Flow cytometry. hFLSCs expressed the markers of stromal cells such as CD105, CD29, CD90, and CD44, but not hematopoietic markers CD34 and CD45. The expression of EPO was analyzed by RT-PCR ( E ) and Western blot ( F ). ( G ) Secretory volume of EPO protein was detected by ELISA. (1: hFLSCs without transfection; 2: hFLSCs transfected with empty vector; 3: hFLSCs transfected with pBPLV-EPO). ( H ) Cytokines expression of EPO/hFLSCs. (1: DL marker 2000; 2: EPOR; 3: SCF; 4: SDF1; 5: IL-6).

    Journal: Cellular Reprogramming

    Article Title: Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells

    doi: 10.1089/cell.2011.0013

    Figure Lengend Snippet: Morphology and identification of EPO overexpressing hFLSCs. ( A ) The of human fetal liver stromal cells (hFLSCs) show typical fibroblast morphology(scale bar=100 μm). ( B ) The growth curve of hFLSCs expressing EPO. The status of growth of cells indicated little difference between hFLSCs expressing ectopic EPO and control FLSCs. ( C ) Expression of enhanced green fluorescence protein (eGFP) in hFLSCs under the fluorescence (scale bar=100 μm). ( D ) Cell phenotype analysis of hFLSCs by Flow cytometry. hFLSCs expressed the markers of stromal cells such as CD105, CD29, CD90, and CD44, but not hematopoietic markers CD34 and CD45. The expression of EPO was analyzed by RT-PCR ( E ) and Western blot ( F ). ( G ) Secretory volume of EPO protein was detected by ELISA. (1: hFLSCs without transfection; 2: hFLSCs transfected with empty vector; 3: hFLSCs transfected with pBPLV-EPO). ( H ) Cytokines expression of EPO/hFLSCs. (1: DL marker 2000; 2: EPOR; 3: SCF; 4: SDF1; 5: IL-6).

    Article Snippet: The trypsinized individual cells were incubated with the FITC-conjugated and PE-conjugated monoclonal antibodies: antihuman CD29, antihuman CD105, antihuman CD44, antihuman CD90, antihuman CD34, and antihuman CD45 (BD Biosciences, San Jose, CA) at 4°C for 30 min. Then the cells were washed three times with PBS and analyzed by flow cytometry analysis using the FACSCalibur (Becton-Dickinson, Mountain View, CA).

    Techniques: Expressing, Fluorescence, Flow Cytometry, Cytometry, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection, Plasmid Preparation, Marker