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Becton Dickinson flow cytometry analysis
In vivo genome editing using the DOX-inducible sgRNA expression construct. ( a ) Schematic diagram of testing the DOX-inducible sgRNA expression vector in bone marrow chimeric mice. ( b ) Representative flow <t>cytometry</t> histograms showing DOX-inducible Cd44 knockout in splenic cells. (D) DOX-inducible Cd44 knockout in CD11b+, CD11c + and CD19+ cells in spleen. ( d ) DOX-inducible Cd44 knockout in CD11b + and CD19+ cells in bone marrow. ( e ) DOX-inducible Cd44 knockout in CD11b + and CD19+ cells in peripheral blood. Individual animals and mean ( c - e ) are shown with five mice per group. P values were derived from t tests: * P
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1) Product Images from "Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening"

Article Title: Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening

Journal: BMC Genomics

doi: 10.1186/s12864-019-5601-9

In vivo genome editing using the DOX-inducible sgRNA expression construct. ( a ) Schematic diagram of testing the DOX-inducible sgRNA expression vector in bone marrow chimeric mice. ( b ) Representative flow cytometry histograms showing DOX-inducible Cd44 knockout in splenic cells. (D) DOX-inducible Cd44 knockout in CD11b+, CD11c + and CD19+ cells in spleen. ( d ) DOX-inducible Cd44 knockout in CD11b + and CD19+ cells in bone marrow. ( e ) DOX-inducible Cd44 knockout in CD11b + and CD19+ cells in peripheral blood. Individual animals and mean ( c - e ) are shown with five mice per group. P values were derived from t tests: * P
Figure Legend Snippet: In vivo genome editing using the DOX-inducible sgRNA expression construct. ( a ) Schematic diagram of testing the DOX-inducible sgRNA expression vector in bone marrow chimeric mice. ( b ) Representative flow cytometry histograms showing DOX-inducible Cd44 knockout in splenic cells. (D) DOX-inducible Cd44 knockout in CD11b+, CD11c + and CD19+ cells in spleen. ( d ) DOX-inducible Cd44 knockout in CD11b + and CD19+ cells in bone marrow. ( e ) DOX-inducible Cd44 knockout in CD11b + and CD19+ cells in peripheral blood. Individual animals and mean ( c - e ) are shown with five mice per group. P values were derived from t tests: * P

Techniques Used: In Vivo, Expressing, Construct, Plasmid Preparation, Mouse Assay, Flow Cytometry, Cytometry, Knock-Out, Derivative Assay

Design and evaluation of drug-inducible sgRNA expression vectors . ( a ) Schematic for drug-inducible sgRNA expression vectors. Cas9 is constitutively expressed in the cells. EGFP reporter gene is used for the quantification of genome editing activity. PAC encodes puromycin N-acetyltransferase. ( b ) Representative flow cytometry histograms showing dose-dependent inducible EGFP knockout in MC-38 for tet - (left) and lac - (right) systems. ( c ) Evaluation of background activity and drug inducible gene knockout efficiency of the inducible sgRNA expression vectors in multiple cell lines. Data represent mean ± SD ( n = 3). P values were derived from t tests: * P
Figure Legend Snippet: Design and evaluation of drug-inducible sgRNA expression vectors . ( a ) Schematic for drug-inducible sgRNA expression vectors. Cas9 is constitutively expressed in the cells. EGFP reporter gene is used for the quantification of genome editing activity. PAC encodes puromycin N-acetyltransferase. ( b ) Representative flow cytometry histograms showing dose-dependent inducible EGFP knockout in MC-38 for tet - (left) and lac - (right) systems. ( c ) Evaluation of background activity and drug inducible gene knockout efficiency of the inducible sgRNA expression vectors in multiple cell lines. Data represent mean ± SD ( n = 3). P values were derived from t tests: * P

Techniques Used: Expressing, Activity Assay, Flow Cytometry, Cytometry, Knock-Out, Gene Knockout, Derivative Assay

2) Product Images from "Revealing the alternative promoter usage of SAF/MAZ gene by bichromatic fluorescent reporter construct"

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

Journal: Bioscience Reports

doi: 10.1042/BSR20171668

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
Figure Legend 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

Techniques Used: 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.
Figure Legend 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.

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

3) Product Images from "Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice"

Article Title: Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice

Journal: Scientific Reports

doi: 10.1038/s41598-019-41805-x

Immunophenotypic change and loss of insertion sites of erythroleukemia following transplantation and cell culture models. ( a ) Flow cytometry results from primary leukemia (top), transplanted leukemia (middle), and cell culture (bottom). The abnormal erythroblasts are CD45 dim/low side scatter/high CD71/variable CD117/variable Ter119 (red). ( b ) Loss of insertion sites following cell culture. Number of total insertion sites and common insertion site (CIS) identified in primary tumor (blue and green) and cell lines (red and purple) in five separate tumors and derived cell lines. ( c ) Quantification of inserts and CIS in tumor-derived cell lines.
Figure Legend Snippet: Immunophenotypic change and loss of insertion sites of erythroleukemia following transplantation and cell culture models. ( a ) Flow cytometry results from primary leukemia (top), transplanted leukemia (middle), and cell culture (bottom). The abnormal erythroblasts are CD45 dim/low side scatter/high CD71/variable CD117/variable Ter119 (red). ( b ) Loss of insertion sites following cell culture. Number of total insertion sites and common insertion site (CIS) identified in primary tumor (blue and green) and cell lines (red and purple) in five separate tumors and derived cell lines. ( c ) Quantification of inserts and CIS in tumor-derived cell lines.

Techniques Used: Transplantation Assay, Cell Culture, Flow Cytometry, Cytometry, Derivative Assay

Breeding scheme, survival, and tumor spectrum of Mx-1 sleeping beauty mice. ( a ) Breeding scheme for transposon mutagenesis in cyclin ET74AT393A and wild-type mice. Cyclin ET74AT393A or wild-type mice homozygous for both a T2/Onc transposon array and Cre recombinase-inducible SB transposase allele (T2/Onc2/T2/Onc2; RosaSBLSL/RosaSBLSL) were mated with cyclin ET74AT393A mice heterozygous for Mx-1 Cre to generate cyclin ET74AT393A or wild-type mice with T2/Onc transposon array and Mx-1 Cre recombinase. ( b ) Kaplan Myer survival curve of SB-Cyclin ET74AT393A and SB-wild-type mice following induction of Mx-1 Cre and transpose expression by poly I/C injection. Time indicates intervention due to illness related to hematologic malignancy. Induction of malignancy was completely penetrant in both backgrounds with a median survival of 11.9 weeks post-injection for wild-type and 12.9 weeks for cyclin ET74AT393A mice (p = 0.08). ( c ) Spectrum of hematologic neoplasms that arise in Mx-1 Sleeping beauty mice. Diagnosis was made by a combination of histology and flow cytometry (see text).
Figure Legend Snippet: Breeding scheme, survival, and tumor spectrum of Mx-1 sleeping beauty mice. ( a ) Breeding scheme for transposon mutagenesis in cyclin ET74AT393A and wild-type mice. Cyclin ET74AT393A or wild-type mice homozygous for both a T2/Onc transposon array and Cre recombinase-inducible SB transposase allele (T2/Onc2/T2/Onc2; RosaSBLSL/RosaSBLSL) were mated with cyclin ET74AT393A mice heterozygous for Mx-1 Cre to generate cyclin ET74AT393A or wild-type mice with T2/Onc transposon array and Mx-1 Cre recombinase. ( b ) Kaplan Myer survival curve of SB-Cyclin ET74AT393A and SB-wild-type mice following induction of Mx-1 Cre and transpose expression by poly I/C injection. Time indicates intervention due to illness related to hematologic malignancy. Induction of malignancy was completely penetrant in both backgrounds with a median survival of 11.9 weeks post-injection for wild-type and 12.9 weeks for cyclin ET74AT393A mice (p = 0.08). ( c ) Spectrum of hematologic neoplasms that arise in Mx-1 Sleeping beauty mice. Diagnosis was made by a combination of histology and flow cytometry (see text).

Techniques Used: Mouse Assay, Mutagenesis, Expressing, Injection, Flow Cytometry, Cytometry

4) Product Images from "Delphinidin, an active compound of red wine, inhibits endothelial cell apoptosis via nitric oxide pathway and regulation of calcium homeostasis"

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

Journal: British Journal of Pharmacology

doi: 10.1038/sj.bjp.0705347

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.
Figure Legend 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.

Techniques Used: Staining, Flow Cytometry, Cytometry, Light Microscopy

5) Product Images from "Human Fetal Liver Stromal Cells Expressing Erythropoietin Promote Hematopoietic Development from Human Embryonic Stem Cells"

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

Journal: Cellular Reprogramming

doi: 10.1089/cell.2011.0013

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.
Figure Legend 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.

Techniques Used: 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).
Figure Legend 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).

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

6) Product Images from "Interleukin 17 Receptor A Modulates Monocyte Subsets and Macrophage Generation In Vivo"

Article Title: Interleukin 17 Receptor A Modulates Monocyte Subsets and Macrophage Generation In Vivo

Journal: PLoS ONE

doi: 10.1371/journal.pone.0085461

Decreased Gr1 low monocyte counts in IL-17 receptor A deficient mice. ( A,B ) Peripheral blood total white cell (A) and monocyte (B) counts were assessed by an automated analyzer in wild type (wt) and Interleukin-17 receptor A-deficient ( Il17ra -/- ) mice (n = 9–12). ( C–E ) Gr1 high and Gr1 low monocyte subgroups were analyzed by flow cytometry after gating for CD11b + CD115 + events (example in C, D: proportion of Gr1 high monocytes and E: absolute concentrations in wt and Il17ra -/- mice, n = 9–12, t-tests).
Figure Legend Snippet: Decreased Gr1 low monocyte counts in IL-17 receptor A deficient mice. ( A,B ) Peripheral blood total white cell (A) and monocyte (B) counts were assessed by an automated analyzer in wild type (wt) and Interleukin-17 receptor A-deficient ( Il17ra -/- ) mice (n = 9–12). ( C–E ) Gr1 high and Gr1 low monocyte subgroups were analyzed by flow cytometry after gating for CD11b + CD115 + events (example in C, D: proportion of Gr1 high monocytes and E: absolute concentrations in wt and Il17ra -/- mice, n = 9–12, t-tests).

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

Homeostatic tissue macrophages are altered without IL-17 receptor. ( A–D ) Monocytes, macrophages and dendritic cells from mixed bone marrow chimeric wt/ Il17ra -/- mice were analyzed by flow cytometry after enzymatic digestion of lungs (examples in A, statistical analysis in B), in spleen (C) and peritoneal cavity (D) (n = 8 and 2 independent transplantations, paired t-tests).
Figure Legend Snippet: Homeostatic tissue macrophages are altered without IL-17 receptor. ( A–D ) Monocytes, macrophages and dendritic cells from mixed bone marrow chimeric wt/ Il17ra -/- mice were analyzed by flow cytometry after enzymatic digestion of lungs (examples in A, statistical analysis in B), in spleen (C) and peritoneal cavity (D) (n = 8 and 2 independent transplantations, paired t-tests).

Techniques Used: Mouse Assay, Flow Cytometry, Cytometry

In peritonitis, Il17ra -/- monocyte recruitment is sustained, but macrophage phenotype changed. ( A,B ) Peritonitis was induced in mixed bone marrow chimeric wt/ Il17ra -/- mice. Peripheral blood (at start and end of the experiment) and peritoneal neutrophils (PMN, CD11b + CD115 − Gr1 high , A) and monocytes (CD11b + CD115 + , B) were analyzed by flow cytometry after 10 h (n = 4, Bonferroni after ANOVA). ( C ) At day 3, intraperitoneal myeloid CD11b + cells were analyzed and compared to peripheral blood monocytes at start and end of the experiment (n = 4–6, 2 independent transplantations, Bonferroni after ANOVA). ( D ) CD11c expression on wt and Il17ra -/- myeloid (CD11b + ) cells was compared in lung, spleen, resting and inflamed peritoneal cavity of mixed bone marrow chimeric wt/ Il17ra -/- mice (n = 4–8, paired t-tests).
Figure Legend Snippet: In peritonitis, Il17ra -/- monocyte recruitment is sustained, but macrophage phenotype changed. ( A,B ) Peritonitis was induced in mixed bone marrow chimeric wt/ Il17ra -/- mice. Peripheral blood (at start and end of the experiment) and peritoneal neutrophils (PMN, CD11b + CD115 − Gr1 high , A) and monocytes (CD11b + CD115 + , B) were analyzed by flow cytometry after 10 h (n = 4, Bonferroni after ANOVA). ( C ) At day 3, intraperitoneal myeloid CD11b + cells were analyzed and compared to peripheral blood monocytes at start and end of the experiment (n = 4–6, 2 independent transplantations, Bonferroni after ANOVA). ( D ) CD11c expression on wt and Il17ra -/- myeloid (CD11b + ) cells was compared in lung, spleen, resting and inflamed peritoneal cavity of mixed bone marrow chimeric wt/ Il17ra -/- mice (n = 4–8, paired t-tests).

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

7) Product Images from "A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity"

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0069405

GIFT9 stimulates the growth of BMMCs. ( A ) Flow cytometry analysis of BMMCs. ( B ) MTT assay of BMMCs after 5 days of culture with GMCSF/IL9 or GIFT9. *: P
Figure Legend Snippet: GIFT9 stimulates the growth of BMMCs. ( A ) Flow cytometry analysis of BMMCs. ( B ) MTT assay of BMMCs after 5 days of culture with GMCSF/IL9 or GIFT9. *: P

Techniques Used: Flow Cytometry, Cytometry, MTT Assay

8) Product Images from "Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis"

Article Title: Adoptive Transfer of Regulatory T Cells Protects against Coxsackievirus B3-Induced Cardiac Fibrosis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0074955

Treg frequency has negative correlation with severity of cardiac fibrosis. Groups of 20 mice were infected with 10 TCID 50 dose of CVB3, 1,2,3,4 wks after infection, spleen and myocardial infiltrated cells were subjected to, flow cytometry protocol and stained for PerCP-CD4 and PE-Foxp3. (A) Frequencies of peripheral and myocardial Tregs were shown, graph representative of three independent experiments. ( B ) Relative numbers of Treg frequency. Data. represent as mean ±SEM. *, P
Figure Legend Snippet: Treg frequency has negative correlation with severity of cardiac fibrosis. Groups of 20 mice were infected with 10 TCID 50 dose of CVB3, 1,2,3,4 wks after infection, spleen and myocardial infiltrated cells were subjected to, flow cytometry protocol and stained for PerCP-CD4 and PE-Foxp3. (A) Frequencies of peripheral and myocardial Tregs were shown, graph representative of three independent experiments. ( B ) Relative numbers of Treg frequency. Data. represent as mean ±SEM. *, P

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

9) Product Images from "Impaired Local Production of Proresolving Lipid Mediators in Obesity and 17-HDHA as a Potential Treatment for Obesity-Associated Inflammation"

Article Title: Impaired Local Production of Proresolving Lipid Mediators in Obesity and 17-HDHA as a Potential Treatment for Obesity-Associated Inflammation

Journal: Diabetes

doi: 10.2337/db12-0828

17-HDHA significantly decreases obesity-associated adipose tissue inflammation. HF-fed WT mice were treated with DHA, 17-HDHA, or vehicle (VE) control via intraperitoneal injection every 12 h for 8 days. 17-HDHA treatment reduced mRNA expression of the inflammatory genes for MCP-1 ( Ccl2 ) ( A ), TNF-a ( Tnf ) ( B ), IL-6 ( Il6 ) ( C ), OPN ( Spp1 ) ( D ), and NF-κB ( Nfkb1 ) ( E ) in gonadal adipose tissue of WT HF animals compared with VE-treated control group ( n = 12–14 animals per group). ( F ) and ( G ) Immunoblot analysis and quantification of IκBα in gonadal adipose tissue of WT HF animals after VE and 17-HDHA treatment. The diagram shows means of the chemiluminescence intensity ratios from IκBα vs. β-tubulin, which was used as the loading control ( G ) ( n = 6 animals per group). ( H ) Representative images of CLS formation in gonadal adipose tissue (scale bar = 50 µm). ( I ) Number of CLS counts per 100 adipocytes in gonadal adipose tissue after VE, DHA, or 17-HDHA treatment ( n = 5–6 animals per group). ( J ) Flow cytometry analysis of the CD11c + CD206 − -to-CD11c − CD206 + ratio of adipose tissue macrophages (F4/80 + cells) obtained from stromal vascular fractions after DHA and 17-HDHA treatment compared with VE control ( n = 5–6 animals per group). For statistical analysis, DHA- and 17-HDHA–treated groups were compared with the VE-treated control group. All data are mean ± SEM. # P
Figure Legend Snippet: 17-HDHA significantly decreases obesity-associated adipose tissue inflammation. HF-fed WT mice were treated with DHA, 17-HDHA, or vehicle (VE) control via intraperitoneal injection every 12 h for 8 days. 17-HDHA treatment reduced mRNA expression of the inflammatory genes for MCP-1 ( Ccl2 ) ( A ), TNF-a ( Tnf ) ( B ), IL-6 ( Il6 ) ( C ), OPN ( Spp1 ) ( D ), and NF-κB ( Nfkb1 ) ( E ) in gonadal adipose tissue of WT HF animals compared with VE-treated control group ( n = 12–14 animals per group). ( F ) and ( G ) Immunoblot analysis and quantification of IκBα in gonadal adipose tissue of WT HF animals after VE and 17-HDHA treatment. The diagram shows means of the chemiluminescence intensity ratios from IκBα vs. β-tubulin, which was used as the loading control ( G ) ( n = 6 animals per group). ( H ) Representative images of CLS formation in gonadal adipose tissue (scale bar = 50 µm). ( I ) Number of CLS counts per 100 adipocytes in gonadal adipose tissue after VE, DHA, or 17-HDHA treatment ( n = 5–6 animals per group). ( J ) Flow cytometry analysis of the CD11c + CD206 − -to-CD11c − CD206 + ratio of adipose tissue macrophages (F4/80 + cells) obtained from stromal vascular fractions after DHA and 17-HDHA treatment compared with VE control ( n = 5–6 animals per group). For statistical analysis, DHA- and 17-HDHA–treated groups were compared with the VE-treated control group. All data are mean ± SEM. # P

Techniques Used: Mouse Assay, Injection, Expressing, Flow Cytometry, Cytometry

Dietary n-3 PUFA treatment attenuates adipose tissue inflammation and improves insulin sensitivity. Lean ( db/+) mice were fed an LF diet and obese ( db/db ) animals were fed an LF diet or three different isocaloric HF diets: 1 ) HF/S diet rich in saturated/monounsaturated fatty acids; 2 ) HF/6 diet rich in n-6 PUFA; and 3 ) HF/S3 (HF/S diet supplemented with n-3 PUFA) for 6 weeks. Gonadal adipose tissue expression of the genes for macrophage marker F4/80 ( Emr1 ) ( A ) and MCP-1 ( Ccl2 ) ( B ) after dietary treatment was analyzed ( n = 10 animals per group). ( C ) CLS formation, a hallmark of obesity-associated inflammation, was assessed by MAC-2 staining, and the number of CLS in gonadal adipose tissue was calculated per 100 adipocytes ( n = 5 animals per group). ( D ) Flow cytometry analysis of the CD11c + CD206 − -to-CD11c − CD206 + ratio of adipose tissue macrophages (F4/80 + cells) obtained from stromal vascular fractions of the db/+ LF control and db/db animals after dietary treatment with indicated diets ( n = 8 animals per group). Expression of the genes for PPARγ ( Pparg ) ( E ), adiponectin ( Adipoq ) ( F ), and GLUT-4 ( Slc2a4 ) ( G ) after dietary treatment in gonadal adipose tissue ( n = 10 animals per group). ( H and I ) Insulin sensitivity was determined in db/db mice after LF control, HF/S, HF/6, or HF/S3 diet. Blood glucose was measured before and 30, 60, 90, and 120 min after intraperitoneal injection of insulin (2.0 units/kg body weight; n = 7–9 animals per group), and the area under the curve was calculated ( n = 7–9 animals per group). For statistical analysis, db/db mice were compared with those fed the HF/S diet. All data are mean ± SEM. # P = 0.067; * P
Figure Legend Snippet: Dietary n-3 PUFA treatment attenuates adipose tissue inflammation and improves insulin sensitivity. Lean ( db/+) mice were fed an LF diet and obese ( db/db ) animals were fed an LF diet or three different isocaloric HF diets: 1 ) HF/S diet rich in saturated/monounsaturated fatty acids; 2 ) HF/6 diet rich in n-6 PUFA; and 3 ) HF/S3 (HF/S diet supplemented with n-3 PUFA) for 6 weeks. Gonadal adipose tissue expression of the genes for macrophage marker F4/80 ( Emr1 ) ( A ) and MCP-1 ( Ccl2 ) ( B ) after dietary treatment was analyzed ( n = 10 animals per group). ( C ) CLS formation, a hallmark of obesity-associated inflammation, was assessed by MAC-2 staining, and the number of CLS in gonadal adipose tissue was calculated per 100 adipocytes ( n = 5 animals per group). ( D ) Flow cytometry analysis of the CD11c + CD206 − -to-CD11c − CD206 + ratio of adipose tissue macrophages (F4/80 + cells) obtained from stromal vascular fractions of the db/+ LF control and db/db animals after dietary treatment with indicated diets ( n = 8 animals per group). Expression of the genes for PPARγ ( Pparg ) ( E ), adiponectin ( Adipoq ) ( F ), and GLUT-4 ( Slc2a4 ) ( G ) after dietary treatment in gonadal adipose tissue ( n = 10 animals per group). ( H and I ) Insulin sensitivity was determined in db/db mice after LF control, HF/S, HF/6, or HF/S3 diet. Blood glucose was measured before and 30, 60, 90, and 120 min after intraperitoneal injection of insulin (2.0 units/kg body weight; n = 7–9 animals per group), and the area under the curve was calculated ( n = 7–9 animals per group). For statistical analysis, db/db mice were compared with those fed the HF/S diet. All data are mean ± SEM. # P = 0.067; * P

Techniques Used: Mouse Assay, Expressing, Marker, Staining, Flow Cytometry, Cytometry, Injection

10) Product Images from "Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM"

Article Title: Variant allele frequency enrichment analysis in vitro reveals sonic hedgehog pathway to impede sustained temozolomide response in GBM

Journal: Scientific Reports

doi: 10.1038/srep07915

Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere. a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). ( ✶ p-value
Figure Legend Snippet: Flow cytometry analysis of annexin-V and propidium iodide (PI) staining of apoptotic cells following vismodegib (50 μM) and TMZ (50 μM) treatment to B0048 neurosphere. a), DMSO-treated control, b), TMZ treatment alone, c) vismodegib treatment alone, d), TMZ treatment along with vismodegib treatment and e), showing % of apoptotic cells (annexin-V positive + PI positive + annexin-V and PI double positive cells). ( ✶ p-value

Techniques Used: Flow Cytometry, Cytometry, Staining

11) Product Images from "Privatization of Biofilm Matrix in Structurally Heterogeneous Biofilms"

Article Title: Privatization of Biofilm Matrix in Structurally Heterogeneous Biofilms

Journal: mSystems

doi: 10.1128/mSystems.00425-20

Expression of matrix genes in robust and fragile fractions of the biofilm. Flow cytometry analysis showing average ( n = 3) distributions of fluorescence intensities of mechanically disrupted P eps -gfp (A) and P tapA -gfp (B) reporter strains after 24, 36, and 48 h. The blue histogram represents the robust fraction, while the yellow graph represents the fragile fraction; the gray graph depicts nonlabeled cells. Data for nonlabeled control were acquired for 48-h-old pellicle and integrated into the upper left histograms of left and right panels, as a red overlay. AU indicates arbitrary units.
Figure Legend Snippet: Expression of matrix genes in robust and fragile fractions of the biofilm. Flow cytometry analysis showing average ( n = 3) distributions of fluorescence intensities of mechanically disrupted P eps -gfp (A) and P tapA -gfp (B) reporter strains after 24, 36, and 48 h. The blue histogram represents the robust fraction, while the yellow graph represents the fragile fraction; the gray graph depicts nonlabeled cells. Data for nonlabeled control were acquired for 48-h-old pellicle and integrated into the upper left histograms of left and right panels, as a red overlay. AU indicates arbitrary units.

Techniques Used: Expressing, Flow Cytometry, Fluorescence

Changes in matrix gene expression during biofilm development assessed by flow cytometry. Flow cytometry analysis showing distributions of fluorescence intensities of GFP-based transcriptional reporters for epsA-epsO (left) and tapA-sipW-tasA (right) at various time points throughout biofilm development. Histograms obtained for all biological replicates ( n = 3) are overlaid for each time point. Data where distribution of matrix gene expression was unimodal (P tapA -GFP, 20 h) are marked with a yellow background. Significant shifts of mean expression level in each subpopulation were indicated by dashed lines and asterisks. Significant changes in relative size of subpopulation with low- and high-matrix gene expression were shown as arrows (pointing toward shift direction) and asterisks. For changes in mean expression and subpopulation relative size, only significant differences between 2 neighboring time points were depicted on the image. Data for nonlabeled control were acquired for 48-h-old pellicle and integrated into the corresponding histograms as a red overlay. AU indicates arbitrary units.
Figure Legend Snippet: Changes in matrix gene expression during biofilm development assessed by flow cytometry. Flow cytometry analysis showing distributions of fluorescence intensities of GFP-based transcriptional reporters for epsA-epsO (left) and tapA-sipW-tasA (right) at various time points throughout biofilm development. Histograms obtained for all biological replicates ( n = 3) are overlaid for each time point. Data where distribution of matrix gene expression was unimodal (P tapA -GFP, 20 h) are marked with a yellow background. Significant shifts of mean expression level in each subpopulation were indicated by dashed lines and asterisks. Significant changes in relative size of subpopulation with low- and high-matrix gene expression were shown as arrows (pointing toward shift direction) and asterisks. For changes in mean expression and subpopulation relative size, only significant differences between 2 neighboring time points were depicted on the image. Data for nonlabeled control were acquired for 48-h-old pellicle and integrated into the corresponding histograms as a red overlay. AU indicates arbitrary units.

Techniques Used: Expressing, Flow Cytometry, Fluorescence

12) Product Images from "Metformin Synergistically Enhanced the Antitumor Activity of Celecoxib in Human Non-Small Cell Lung Cancer Cells"

Article Title: Metformin Synergistically Enhanced the Antitumor Activity of Celecoxib in Human Non-Small Cell Lung Cancer Cells

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2020.01094

Combined treatment with celecoxib and metformin increases reactive oxygen species and DNA damage in NSCLC cells. (A) Detection of ROS accumulation in A549 and H1299 cells by fluorescent probe DCFH-DA. The change of fluorescence intensity was observed by flow Cytometry. (B) Detection of ROS accumulation in A549 and H1299 cells by fluorescent probe DCFH-DA. The change of fluorescence intensity was observed by Fluorescence microscope. (C) Immunofluorescence staining of γ-H2AX in A549 cells treated with 10 mM metformin and/or 25 μM celecoxib. (D) Effects of celecoxib and metformin on γ-H2AX, p-ATM, p-CHK2, and p53 protein expression. A549 cells were treated with 10 mM metformin and/or 25 μM celecoxib for 48 h, and then harvested for WB analysis using indicated antibodies. The level of actin served as the loading control. Band intensities were calculated using software Image J. Relative intensities are also shown. Data represent mean values of triplicate samples. Data are represented as mean ± SD. * p
Figure Legend Snippet: Combined treatment with celecoxib and metformin increases reactive oxygen species and DNA damage in NSCLC cells. (A) Detection of ROS accumulation in A549 and H1299 cells by fluorescent probe DCFH-DA. The change of fluorescence intensity was observed by flow Cytometry. (B) Detection of ROS accumulation in A549 and H1299 cells by fluorescent probe DCFH-DA. The change of fluorescence intensity was observed by Fluorescence microscope. (C) Immunofluorescence staining of γ-H2AX in A549 cells treated with 10 mM metformin and/or 25 μM celecoxib. (D) Effects of celecoxib and metformin on γ-H2AX, p-ATM, p-CHK2, and p53 protein expression. A549 cells were treated with 10 mM metformin and/or 25 μM celecoxib for 48 h, and then harvested for WB analysis using indicated antibodies. The level of actin served as the loading control. Band intensities were calculated using software Image J. Relative intensities are also shown. Data represent mean values of triplicate samples. Data are represented as mean ± SD. * p

Techniques Used: Fluorescence, Flow Cytometry, Microscopy, Immunofluorescence, Staining, Expressing, Western Blot, Software

13) Product Images from "Compound Prunetin Induces Cell Death in Gastric Cancer Cell with Potent Anti-Proliferative Properties: In Vitro Assay, Molecular Docking, Dynamics, and ADMET Studies"

Article Title: Compound Prunetin Induces Cell Death in Gastric Cancer Cell with Potent Anti-Proliferative Properties: In Vitro Assay, Molecular Docking, Dynamics, and ADMET Studies

Journal: Biomolecules

doi: 10.3390/biom10071086

Analysis of cell cycle by flow cytometry. ( a ) The effect of PRU on cell cycle arrest was examined by flow cytometry analysis, AGS cells were treated with PRU at indicated concentrations (20, 40, and 80 μM) for 24 h. Followed by propidium iodide (PI) staining and subjected to flow cytometer analysis. ( b ) The population of cells identified on different phases of the cell cycle were represented graphically. Values are given as the mean ± standard error of the mean (SEM) of three independent experiments. * p
Figure Legend Snippet: Analysis of cell cycle by flow cytometry. ( a ) The effect of PRU on cell cycle arrest was examined by flow cytometry analysis, AGS cells were treated with PRU at indicated concentrations (20, 40, and 80 μM) for 24 h. Followed by propidium iodide (PI) staining and subjected to flow cytometer analysis. ( b ) The population of cells identified on different phases of the cell cycle were represented graphically. Values are given as the mean ± standard error of the mean (SEM) of three independent experiments. * p

Techniques Used: Flow Cytometry, Staining

Effect of prunetin on necroptosis related cell death. ( a ) The effect of PRU on cell cycle arrest was examined by flow cytometry analysis, AGS cells were treated with PRU at indicated concentrations (20, 40, and 80 μM) for 24 h. Followed by allophycocyanin (APC)/annexin V and propidium iodide (PI) double-staining was performed which was analyzed by flow cytometry. ( b ) The population of cells identified on the different state of cell death were represented graphically. ( c ) Cell viability assay performed on PRU treated AGS cells with and without co-treatment with necrostatin-1 inhibitor (0.1 mM). ( d ) Hematoxylin staining on PRU treated AGS cells with visible necroptic morphology such as cell swelling, visible disruption of cell organelles, and formation of vacuoles. Values are given as the mean ± standard error of the mean (SEM) of three independent experiments. * p
Figure Legend Snippet: Effect of prunetin on necroptosis related cell death. ( a ) The effect of PRU on cell cycle arrest was examined by flow cytometry analysis, AGS cells were treated with PRU at indicated concentrations (20, 40, and 80 μM) for 24 h. Followed by allophycocyanin (APC)/annexin V and propidium iodide (PI) double-staining was performed which was analyzed by flow cytometry. ( b ) The population of cells identified on the different state of cell death were represented graphically. ( c ) Cell viability assay performed on PRU treated AGS cells with and without co-treatment with necrostatin-1 inhibitor (0.1 mM). ( d ) Hematoxylin staining on PRU treated AGS cells with visible necroptic morphology such as cell swelling, visible disruption of cell organelles, and formation of vacuoles. Values are given as the mean ± standard error of the mean (SEM) of three independent experiments. * p

Techniques Used: Flow Cytometry, Double Staining, Viability Assay, Staining

14) Product Images from "Fasting Therapy Contributes to the Improvement of Endothelial Function and Decline in Vascular Injury-Related Markers in Overweight and Obese Individuals via Activating Autophagy of Endothelial Progenitor Cells"

Article Title: Fasting Therapy Contributes to the Improvement of Endothelial Function and Decline in Vascular Injury-Related Markers in Overweight and Obese Individuals via Activating Autophagy of Endothelial Progenitor Cells

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2020/3576030

Identification of EPCs. (a) The morphology of late EPCs (100x magnification). (b) The late EPCs markers labeled by flow cytometry.
Figure Legend Snippet: Identification of EPCs. (a) The morphology of late EPCs (100x magnification). (b) The late EPCs markers labeled by flow cytometry.

Techniques Used: Labeling, Flow Cytometry

15) Product Images from "Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria"

Article Title: Identification of a New Lipoprotein Export Signal in Gram-Negative Bacteria

Journal: mBio

doi: 10.1128/mBio.01232-16

The position of the minimal LES is crucial for its function. (A) wt SiaC and consensus sequence mutant constructs. Amino acids derived from the consensus sequence (green boldface) and point mutations (gray boldface) are indicated. The SiaC constructs are referred to by the boldface numbers shown in panel A in panels B to E. (B) Detection of SiaC by Western blot analysis of total cell extracts of strains expressing the SiaC constructs shown in panel A. MucG expression was monitored as a loading control. (C) Quantification of SiaC surface exposure by flow cytometry of live cells labeled with anti-SiaC serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 3 are indicated (***). The percentage and standard deviation (SD) of stained cells are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on gray background. Values for strains with a statistically significant lower stained population are shown in red ( P ≤ 0.001 compared to the value for the reference construct 3). (D) Immunofluorescence microscopy images of bacteria stained with anti-SiaC serum. Bar, 5 µm. (E) Western blot analysis of total lysates (TL) and outer membrane (OM) fractions of bacteria expressing different SiaC constructs. MucG expression was monitored as a loading control.
Figure Legend Snippet: The position of the minimal LES is crucial for its function. (A) wt SiaC and consensus sequence mutant constructs. Amino acids derived from the consensus sequence (green boldface) and point mutations (gray boldface) are indicated. The SiaC constructs are referred to by the boldface numbers shown in panel A in panels B to E. (B) Detection of SiaC by Western blot analysis of total cell extracts of strains expressing the SiaC constructs shown in panel A. MucG expression was monitored as a loading control. (C) Quantification of SiaC surface exposure by flow cytometry of live cells labeled with anti-SiaC serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 3 are indicated (***). The percentage and standard deviation (SD) of stained cells are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on gray background. Values for strains with a statistically significant lower stained population are shown in red ( P ≤ 0.001 compared to the value for the reference construct 3). (D) Immunofluorescence microscopy images of bacteria stained with anti-SiaC serum. Bar, 5 µm. (E) Western blot analysis of total lysates (TL) and outer membrane (OM) fractions of bacteria expressing different SiaC constructs. MucG expression was monitored as a loading control.

Techniques Used: Sequencing, Mutagenesis, Construct, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Cytometry, Labeling, Fluorescence, Staining, Standard Deviation, Immunofluorescence, Microscopy

B. fragilis and F. johnsoniae LES allow SiaC surface localization. (A) wt SiaC and consensus sequence mutant constructs. Amino acids derived from the B. fragilis or F. johnsoniae consensus sequence (green boldface) and point mutations (gray boldface) are indicated. The SiaC constructs are referred to by the boldface numbers shown in panel A in panels B to D. (B) Detection of SiaC by Western blot analysis of total cell extracts of strains expressing the SiaC constructs shown in panel A. MucG expression was monitored as a loading control. (C) Quantification of SiaC surface exposure by flow cytometry of live cells labeled with anti-SiaC serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 3 are indicated (***). The percentage and standard deviation (SD) of stained cells are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on a gray background. (D) Immunofluorescence microscopy images of bacteria labeled with anti-SiaC serum. Bar, 5 µm.
Figure Legend Snippet: B. fragilis and F. johnsoniae LES allow SiaC surface localization. (A) wt SiaC and consensus sequence mutant constructs. Amino acids derived from the B. fragilis or F. johnsoniae consensus sequence (green boldface) and point mutations (gray boldface) are indicated. The SiaC constructs are referred to by the boldface numbers shown in panel A in panels B to D. (B) Detection of SiaC by Western blot analysis of total cell extracts of strains expressing the SiaC constructs shown in panel A. MucG expression was monitored as a loading control. (C) Quantification of SiaC surface exposure by flow cytometry of live cells labeled with anti-SiaC serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 3 are indicated (***). The percentage and standard deviation (SD) of stained cells are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on a gray background. (D) Immunofluorescence microscopy images of bacteria labeled with anti-SiaC serum. Bar, 5 µm.

Techniques Used: Sequencing, Mutagenesis, Construct, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Cytometry, Labeling, Fluorescence, Staining, Standard Deviation, Immunofluorescence, Microscopy

The LES allows SiaC surface exposure. (A) Wild-type (wt) SiaC and consensus sequence mutant constructs. Amino acids derived from the consensus sequence (green boldface) and point mutations (gray boldface) are indicated. The SiaC constructs are referred to by the boldface numbers shown in panel A in panels B to E. (B) Detection of SiaC by Western blot analysis of total cell extracts of strains expressing the SiaC constructs shown in panel A. Expression of MucG was monitored as a loading control. (C) Detection of SiaC by Western blot analysis of total lysates (TL) and outer membrane (OM) fractions of bacteria expressing different SiaC constructs. Expression of MucG was monitored as a loading control. (D) Quantification of SiaC surface exposure by flow cytometry of live cells labeled with anti-SiaC serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 3 are indicated (***). Values that are not significantly different (n.s) from the value for reference construct 3 are indicated. The percentage of stained cells and standard deviation (SD) are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on a gray background. Values for strains with a statistically significant lower stained population are shown in red ( P ≤ 0.001 compared to reference construct 3). (E) Immunofluorescence microscopy images of bacteria labeled with anti-SiaC serum. Bar, 5 µm.
Figure Legend Snippet: The LES allows SiaC surface exposure. (A) Wild-type (wt) SiaC and consensus sequence mutant constructs. Amino acids derived from the consensus sequence (green boldface) and point mutations (gray boldface) are indicated. The SiaC constructs are referred to by the boldface numbers shown in panel A in panels B to E. (B) Detection of SiaC by Western blot analysis of total cell extracts of strains expressing the SiaC constructs shown in panel A. Expression of MucG was monitored as a loading control. (C) Detection of SiaC by Western blot analysis of total lysates (TL) and outer membrane (OM) fractions of bacteria expressing different SiaC constructs. Expression of MucG was monitored as a loading control. (D) Quantification of SiaC surface exposure by flow cytometry of live cells labeled with anti-SiaC serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 3 are indicated (***). Values that are not significantly different (n.s) from the value for reference construct 3 are indicated. The percentage of stained cells and standard deviation (SD) are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on a gray background. Values for strains with a statistically significant lower stained population are shown in red ( P ≤ 0.001 compared to reference construct 3). (E) Immunofluorescence microscopy images of bacteria labeled with anti-SiaC serum. Bar, 5 µm.

Techniques Used: Sequencing, Mutagenesis, Construct, Derivative Assay, Western Blot, Expressing, Flow Cytometry, Cytometry, Labeling, Fluorescence, Staining, Standard Deviation, Immunofluorescence, Microscopy

MucG LES mutational analysis. (A) wt MucG and mutant constructs. Point mutations are indicated in gray boldface type. The MucG constructs are referred to by the boldface numbers shown in panel A in panels B and C. (B) Detection of MucG by Western blot analysis of total cell extracts of strains expressing the MucG constructs shown in panel A. Expression of SiaC was monitored as a loading control. (C) Quantification of MucG surface exposure by flow cytometry of live cells labeled with anti-MucG serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 1 are indicated (***). Values that are not significantly different (n.s) from the value for reference construct 1 are indicated. The percentage and standard deviation (SD) of stained cells are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on a gray background. Values for strains with a statistically significant lower stained population are shown in red ( P ≤ 0.001 compared to the value for reference construct 1).
Figure Legend Snippet: MucG LES mutational analysis. (A) wt MucG and mutant constructs. Point mutations are indicated in gray boldface type. The MucG constructs are referred to by the boldface numbers shown in panel A in panels B and C. (B) Detection of MucG by Western blot analysis of total cell extracts of strains expressing the MucG constructs shown in panel A. Expression of SiaC was monitored as a loading control. (C) Quantification of MucG surface exposure by flow cytometry of live cells labeled with anti-MucG serum. The fluorescence intensity of stained cells only is shown (NR, not relevant). The averages from at least three independent experiments are shown. Error bars represent 1 standard deviation from the mean. Values that are significantly different ( P ≤ 0.001) from the value for reference construct 1 are indicated (***). Values that are not significantly different (n.s) from the value for reference construct 1 are indicated. The percentage and standard deviation (SD) of stained cells are indicated below the bar graph. Values below the detection limit (≤2.5%) are shown on a gray background. Values for strains with a statistically significant lower stained population are shown in red ( P ≤ 0.001 compared to the value for reference construct 1).

Techniques Used: Mutagenesis, Construct, Western Blot, Expressing, Flow Cytometry, Cytometry, Labeling, Fluorescence, Staining, Standard Deviation

16) Product Images from "A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes"

Article Title: A Defined and Scalable Peptide-Based Platform for the Generation of Human Pluripotent Stem Cell-Derived Astrocytes

Journal: ACS Biomaterials Science & Engineering

doi: 10.1021/acsbiomaterials.0c00067

Analysis of β-amyloid (Aβ) uptake in astrocytes. Flow cytometry analysis of Aβ internalization in untreated (black traces) and Aβ-FITC-treated (green traces) astrocytes.
Figure Legend Snippet: Analysis of β-amyloid (Aβ) uptake in astrocytes. Flow cytometry analysis of Aβ internalization in untreated (black traces) and Aβ-FITC-treated (green traces) astrocytes.

Techniques Used: Flow Cytometry

Characterization of cryopreserved astrocytes generated on VDP. (A) Representative phase contrast images of pre- and post-cryopreserved astrocytes generated on VDP-coated surfaces. (B) Representative immunofluorescent images of GFAP (left panels) and S100β (right panels) in pre- and post-cryopreserved astrocytes. (C) Representative flow cytometry plots of propidium iodide staining (left panel) and S100β expression (right panel) in pre- and postcryopreserved astrocytes. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 1 as appropriate. (D) Measurement of secreted ApoE in pre- and post-cryopreserved astrocytes cultured on VDP surfaces. N.S. = not statistically significant, Student’s t test. (E) Profile of pro- and anti-inflammatory cytokines in pre- and post-cryopreserved astrocytes cultured under basal conditions. (F) Measurement of upregulated cytokines in pre- and post-cryopreserved astrocytes after treatment with LPS. Data is shown as fold-change increase in cytokine release compared to untreated astrocytes. (G) Measurement of changes (Δ F / F ) in fluorescence of calcium indicator (Fluo-4) in single pre- and post-cryopreserved astrocytes. Inset images of Fluo-4 stained astrocytes are shown at the indicated time points.
Figure Legend Snippet: Characterization of cryopreserved astrocytes generated on VDP. (A) Representative phase contrast images of pre- and post-cryopreserved astrocytes generated on VDP-coated surfaces. (B) Representative immunofluorescent images of GFAP (left panels) and S100β (right panels) in pre- and post-cryopreserved astrocytes. (C) Representative flow cytometry plots of propidium iodide staining (left panel) and S100β expression (right panel) in pre- and postcryopreserved astrocytes. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 1 as appropriate. (D) Measurement of secreted ApoE in pre- and post-cryopreserved astrocytes cultured on VDP surfaces. N.S. = not statistically significant, Student’s t test. (E) Profile of pro- and anti-inflammatory cytokines in pre- and post-cryopreserved astrocytes cultured under basal conditions. (F) Measurement of upregulated cytokines in pre- and post-cryopreserved astrocytes after treatment with LPS. Data is shown as fold-change increase in cytokine release compared to untreated astrocytes. (G) Measurement of changes (Δ F / F ) in fluorescence of calcium indicator (Fluo-4) in single pre- and post-cryopreserved astrocytes. Inset images of Fluo-4 stained astrocytes are shown at the indicated time points.

Techniques Used: Generated, Flow Cytometry, Staining, Expressing, Cell Culture, Fluorescence

Generation of hPSC-derived astrocytes on a completely synthetic peptide substrate. (A) Representative phase contrast images of D50+ hPSC-derived astrocytes generated (scale bar = 200 μm). (B) Representative flow cytometry plots of CD44 expression of D30+ astrocytes on VDP (green traces) and LN (red traces) substrates. Gates were determined using isotype antibody only controls listed in Supplementary Table 1 . (C) Quantitative PCR (qPCR) analysis for expression of astrocyte markers GFAP and VIM in D50+ cultures. Gene expression fold changes were calculated relative to expression levels in undifferentiated hNPCs. (D) Immunofluorescence analysis for expression of GFAP and S100β in D50+ cultures. (E) Representative flow cytometry plots of S100β expression of D50+ astrocytes. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 1 .
Figure Legend Snippet: Generation of hPSC-derived astrocytes on a completely synthetic peptide substrate. (A) Representative phase contrast images of D50+ hPSC-derived astrocytes generated (scale bar = 200 μm). (B) Representative flow cytometry plots of CD44 expression of D30+ astrocytes on VDP (green traces) and LN (red traces) substrates. Gates were determined using isotype antibody only controls listed in Supplementary Table 1 . (C) Quantitative PCR (qPCR) analysis for expression of astrocyte markers GFAP and VIM in D50+ cultures. Gene expression fold changes were calculated relative to expression levels in undifferentiated hNPCs. (D) Immunofluorescence analysis for expression of GFAP and S100β in D50+ cultures. (E) Representative flow cytometry plots of S100β expression of D50+ astrocytes. Gates were determined using isotype or secondary antibody only controls listed in Supplementary Table 1 .

Techniques Used: Derivative Assay, Generated, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence

17) Product Images from "Continuous Genetic Recording with Self-Targeting CRISPR-Cas in Human Cells"

Article Title: Continuous Genetic Recording with Self-Targeting CRISPR-Cas in Human Cells

Journal: bioRxiv

doi: 10.1101/053058

Validating functionality of the Mutation-Based Toggling Reporter (MBTR) system with different indel sizes in the Mutation Detection Region (MDR). We built MBTR constructs with stgRNAs containing indels of different sizes in the MDR (MDR: 0 bp = without an indel, MDR: −1 bp = with a −1 bp indel and MDR: −2 bp = with a −2 bp indel, Constructs 13-15, Table S2). We integrated these constructs into the genome of HEK 293T cells that do not express Cas9 via lentiviral infections at 0.3 MOI. We observed the expected correspondence between indel sizes in the MDR and fluorescence outputs as shown with flow cytometry analysis (top) and fluorescent microscopy (bottom). Also see Fig. 2A .
Figure Legend Snippet: Validating functionality of the Mutation-Based Toggling Reporter (MBTR) system with different indel sizes in the Mutation Detection Region (MDR). We built MBTR constructs with stgRNAs containing indels of different sizes in the MDR (MDR: 0 bp = without an indel, MDR: −1 bp = with a −1 bp indel and MDR: −2 bp = with a −2 bp indel, Constructs 13-15, Table S2). We integrated these constructs into the genome of HEK 293T cells that do not express Cas9 via lentiviral infections at 0.3 MOI. We observed the expected correspondence between indel sizes in the MDR and fluorescence outputs as shown with flow cytometry analysis (top) and fluorescent microscopy (bottom). Also see Fig. 2A .

Techniques Used: Mutagenesis, Construct, Fluorescence, Flow Cytometry, Microscopy

Tracking repetitive and continuous self-targeting activity at the stgRNA locus. ( A ) Schematic of the Mutation-Based Toggling Reporter system (MBTR system) consisting of an stgRNA in the Mutation Detection Region (MDR) or a regular sgRNA target sequence in the MDR region. We illustrate the expected fluorescent readouts of the MBTR system based on different indel sizes in the MDR. In the self-targeting scenario, a U6 promoter driven stgRNA with a 27 nt SDS is embedded between a constitutive human CMV promoter and modified GFP and RFP reporters. RNAP II mediated transcription starts upstream of the U6 promoter. Correct reading frames of each protein relative to the start codon are indicated in the superscript as F1, F2 and F3. Different sizes of indel formation at the stgRNA locus should result in different peptides sequences being translated. When translated in-frame, two ‘self-cleaving’ 2A peptides, P2A and T2A, are designed to cause co-translational ‘cleavage’ of the peptides and release functional fluorescent protein from the nonsense peptides, thus resulting in the appropriate fluorescent signal. The non-self-targeting construct consists of a U6 promoter driving expression of a regular sgRNA, which targets a sequence corresponding to the sgRNA embedded in the MBTR system as the MDR. ( B ) An outline illustrating the double-sorting experiment that tracks repetitive self-targeting activity using the MBTR system. HEK 293T cells stably expressing Cas9 (UBCp-Cas9 cells) were infected with MBTR constructs at low titre so that most of the infected cells had a single copy of the construct. Five days after the initial infection, generation 1 (Gen1) cells were sorted into RFP or GFP positive populations (Gen1:RFP and Gen1:GFP). The genomic DNA was extracted from a portion of the sorted cells. The rest of the sorted cells were allowed to grow to acquire further mutations at the stgRNA loci. The cells initially sorted for RFP or GFP fluorescence (Gen2R and Gen2G) were sorted again seven days after the first sort. The genomic DNA of the sorted cells (Gen2R:RFP, Gen2R:GFP, Gen2G:RFP and Gen2G:GFP) was collected, PCR amplified and Sanger sequenced after bacterial cloning. ( C ) Microscopy analysis and ( D ) flow cytometry data before the 1 st and 2 nd sorting of UBCp-Cas9 cells containing the self-targeting or non-self-targeting MBTR constructs. The white arrows in the microscope images indicate cells that expressed a fluorescent protein different from the one they were sorted for seven days earlier. For the self-targeting MBTR, we found that a significant fraction of cells expressed a fluorescent protein different from the one they were sorted for seven days earlier, indicating that repeated self-targeting mutagenesis that changed the indel sizes of the MDR (at least one mutagenesis event before the 1 st sort and at least one mutagenesis event before the 2 nd sort) occurred at each of the individual stgRNA loci present in the sorted cells. For the non-self-targeting MBTR, we observed minimal fractions of cells that expressed a fluorescent protein different from the one they were sorted for seven days earlier. ( E ) The genomic DNA collected from sorted cells was amplified and cloned into E. coli ; the resulting bacterial colonies were then Sanger sequenced (Methods). A sample of Sanger sequences for the different sorted populations is presented along with their mutation type and the correct reading frame annotated. We observed a high correspondence between the mutated genotype and the observed fluorescent protein expression phenotype. Also see Fig. S2 , S3 .
Figure Legend Snippet: Tracking repetitive and continuous self-targeting activity at the stgRNA locus. ( A ) Schematic of the Mutation-Based Toggling Reporter system (MBTR system) consisting of an stgRNA in the Mutation Detection Region (MDR) or a regular sgRNA target sequence in the MDR region. We illustrate the expected fluorescent readouts of the MBTR system based on different indel sizes in the MDR. In the self-targeting scenario, a U6 promoter driven stgRNA with a 27 nt SDS is embedded between a constitutive human CMV promoter and modified GFP and RFP reporters. RNAP II mediated transcription starts upstream of the U6 promoter. Correct reading frames of each protein relative to the start codon are indicated in the superscript as F1, F2 and F3. Different sizes of indel formation at the stgRNA locus should result in different peptides sequences being translated. When translated in-frame, two ‘self-cleaving’ 2A peptides, P2A and T2A, are designed to cause co-translational ‘cleavage’ of the peptides and release functional fluorescent protein from the nonsense peptides, thus resulting in the appropriate fluorescent signal. The non-self-targeting construct consists of a U6 promoter driving expression of a regular sgRNA, which targets a sequence corresponding to the sgRNA embedded in the MBTR system as the MDR. ( B ) An outline illustrating the double-sorting experiment that tracks repetitive self-targeting activity using the MBTR system. HEK 293T cells stably expressing Cas9 (UBCp-Cas9 cells) were infected with MBTR constructs at low titre so that most of the infected cells had a single copy of the construct. Five days after the initial infection, generation 1 (Gen1) cells were sorted into RFP or GFP positive populations (Gen1:RFP and Gen1:GFP). The genomic DNA was extracted from a portion of the sorted cells. The rest of the sorted cells were allowed to grow to acquire further mutations at the stgRNA loci. The cells initially sorted for RFP or GFP fluorescence (Gen2R and Gen2G) were sorted again seven days after the first sort. The genomic DNA of the sorted cells (Gen2R:RFP, Gen2R:GFP, Gen2G:RFP and Gen2G:GFP) was collected, PCR amplified and Sanger sequenced after bacterial cloning. ( C ) Microscopy analysis and ( D ) flow cytometry data before the 1 st and 2 nd sorting of UBCp-Cas9 cells containing the self-targeting or non-self-targeting MBTR constructs. The white arrows in the microscope images indicate cells that expressed a fluorescent protein different from the one they were sorted for seven days earlier. For the self-targeting MBTR, we found that a significant fraction of cells expressed a fluorescent protein different from the one they were sorted for seven days earlier, indicating that repeated self-targeting mutagenesis that changed the indel sizes of the MDR (at least one mutagenesis event before the 1 st sort and at least one mutagenesis event before the 2 nd sort) occurred at each of the individual stgRNA loci present in the sorted cells. For the non-self-targeting MBTR, we observed minimal fractions of cells that expressed a fluorescent protein different from the one they were sorted for seven days earlier. ( E ) The genomic DNA collected from sorted cells was amplified and cloned into E. coli ; the resulting bacterial colonies were then Sanger sequenced (Methods). A sample of Sanger sequences for the different sorted populations is presented along with their mutation type and the correct reading frame annotated. We observed a high correspondence between the mutated genotype and the observed fluorescent protein expression phenotype. Also see Fig. S2 , S3 .

Techniques Used: Activity Assay, Mutagenesis, Sequencing, Modification, Functional Assay, Construct, Expressing, Stable Transfection, Infection, Fluorescence, Polymerase Chain Reaction, Amplification, Clone Assay, Microscopy, Flow Cytometry

18) Product Images from "A Molecular Mechanism for Probabilistic Bet-hedging and its Role in Viral Latency"

Article Title: A Molecular Mechanism for Probabilistic Bet-hedging and its Role in Viral Latency

Journal: bioRxiv

doi: 10.1101/2020.05.14.096560

The ‘cost’ of high pp71: High virion pp71 levels overcome nuclear exclusion in undifferentiated cells and impede establishment of viral silencing. ( A ) Flow cytometry analysis of undifferentiated NTera2 cells infected with dual-reporter TB40E-IE-mCherry-EYFP. Cells were either mock infected (left), infected with pp71 WT virus (middle) or with pp71 HI virus (right) at MOI=3. ( B ) Quantification of % IE double-positive population for three biological replicates from the flow cytometry data shown in Fig. 3 A (p-values from Student’s t test: ****
Figure Legend Snippet: The ‘cost’ of high pp71: High virion pp71 levels overcome nuclear exclusion in undifferentiated cells and impede establishment of viral silencing. ( A ) Flow cytometry analysis of undifferentiated NTera2 cells infected with dual-reporter TB40E-IE-mCherry-EYFP. Cells were either mock infected (left), infected with pp71 WT virus (middle) or with pp71 HI virus (right) at MOI=3. ( B ) Quantification of % IE double-positive population for three biological replicates from the flow cytometry data shown in Fig. 3 A (p-values from Student’s t test: ****

Techniques Used: Flow Cytometry, Infection

19) Product Images from "PDGFRA Defines the Mesenchymal Stem Cell Kaposi’s Sarcoma Progenitors by Enabling KSHV Oncogenesis in an Angiogenic Environment"

Article Title: PDGFRA Defines the Mesenchymal Stem Cell Kaposi’s Sarcoma Progenitors by Enabling KSHV Oncogenesis in an Angiogenic Environment

Journal: bioRxiv

doi: 10.1101/789826

KSHV infection is only tumorigenic in infected MSC PDGFRA-positive lineagegrown in pro-angiogenic KS-like environment. A) Mouse bone marrow-derived mesenchymal stem cells were stained for PDGFRA (Pα) and SCA-1 (S) expression. Mouse MSC Sca-1-positive, PDGFRA-positive (Pα(+)S) and negative (Pα(-)S) populations were sorted by flow cytometry. B) PDGFRA-positive (Pα(+)S) and PDGFRA-negative (Pα(-)S) MSCs were latently infected with rKSHV.219 and analyzed for GFP expression using a fluorescence microscope. C) Immunofluorescence analysis of KSHV-infected Pα(+)S (K-Pα(+)S) and Pα(-)S (K-Pα(-)S) to evaluate KSHV LANA expression (red), nuclei were counterstained with DAPI (blue). D) Fold-changes in KSHV gene expression between 24 hours KSHV post-infection and after KSHV latency establishment in MSC or KS-like media as determined by RT-qPCR. Triplicates are shown as means ± SD. K-Pα(-)S population on the top and K-Pα(+)S population on the bottom. *P
Figure Legend Snippet: KSHV infection is only tumorigenic in infected MSC PDGFRA-positive lineagegrown in pro-angiogenic KS-like environment. A) Mouse bone marrow-derived mesenchymal stem cells were stained for PDGFRA (Pα) and SCA-1 (S) expression. Mouse MSC Sca-1-positive, PDGFRA-positive (Pα(+)S) and negative (Pα(-)S) populations were sorted by flow cytometry. B) PDGFRA-positive (Pα(+)S) and PDGFRA-negative (Pα(-)S) MSCs were latently infected with rKSHV.219 and analyzed for GFP expression using a fluorescence microscope. C) Immunofluorescence analysis of KSHV-infected Pα(+)S (K-Pα(+)S) and Pα(-)S (K-Pα(-)S) to evaluate KSHV LANA expression (red), nuclei were counterstained with DAPI (blue). D) Fold-changes in KSHV gene expression between 24 hours KSHV post-infection and after KSHV latency establishment in MSC or KS-like media as determined by RT-qPCR. Triplicates are shown as means ± SD. K-Pα(-)S population on the top and K-Pα(+)S population on the bottom. *P

Techniques Used: Infection, Derivative Assay, Staining, Expressing, Flow Cytometry, Fluorescence, Microscopy, Immunofluorescence, Quantitative RT-PCR

20) Product Images from "Etv6/Runx1 Fusion Gene Abrogation Decreases The Oncogenic Potencial Of Tumour Cells In A Preclinical Model Of Acute Lymphoblastic Leukaemia"

Article Title: Etv6/Runx1 Fusion Gene Abrogation Decreases The Oncogenic Potencial Of Tumour Cells In A Preclinical Model Of Acute Lymphoblastic Leukaemia

Journal: bioRxiv

doi: 10.1101/809525

In vitro functional studies after E/R abrogation. (A) Cell cycle distribution of control clones and E/R KO cells at 48 h. (B) CFSE quantification by flow cytometry after 48 in culture. The peak on the right (10 3 ) represents the percentage of cells that have not divided and the left peak (10 2 ) represents the percentage of cells that have divided and therefore diluted their CFSE expression. (C) CFSE expression by flow cytometry of cells co-cultured with MSC cell line HS-5 at 48h. (D) Apoptosis level quantification by PI expression. The figure shows the percentage of PI negative cells (left) and PI positive cells (right) at 48 h. (E) Apoptosis level quantification by PI expression after treatment with Vincristine (1 µM) at 48 h. On the right is represented the mean distribution of control clones (dark grey) and E/R KO clones (grey) of different experiments. All the experiments were carried out by triplicate. * P≤ 0.05 (unpaired t -test).
Figure Legend Snippet: In vitro functional studies after E/R abrogation. (A) Cell cycle distribution of control clones and E/R KO cells at 48 h. (B) CFSE quantification by flow cytometry after 48 in culture. The peak on the right (10 3 ) represents the percentage of cells that have not divided and the left peak (10 2 ) represents the percentage of cells that have divided and therefore diluted their CFSE expression. (C) CFSE expression by flow cytometry of cells co-cultured with MSC cell line HS-5 at 48h. (D) Apoptosis level quantification by PI expression. The figure shows the percentage of PI negative cells (left) and PI positive cells (right) at 48 h. (E) Apoptosis level quantification by PI expression after treatment with Vincristine (1 µM) at 48 h. On the right is represented the mean distribution of control clones (dark grey) and E/R KO clones (grey) of different experiments. All the experiments were carried out by triplicate. * P≤ 0.05 (unpaired t -test).

Techniques Used: In Vitro, Functional Assay, Clone Assay, Flow Cytometry, Expressing, Cell Culture

21) Product Images from "CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer"

Article Title: CX-5461 activates the DNA damage response and demonstrates therapeutic efficacy in high-grade serous ovarian cancer

Journal: Nature Communications

doi: 10.1038/s41467-020-16393-4

CX-5461 has significant therapeutic efficacy in HGSOC- patient-derived xenografts (PDX) models. a Responses observed in post-platinum treated BRCA2-mutant PDX#19 HGSOC-PDX and b PDX #62 with BRCA1 promoter methylation to CX-5461 and olaparib treatment in vivo. Recipient mice bearing the PDX were randomized to treatment with vehicle, 40 mg/kg CX-5461 twice a week, 50 mg/kg olaparib once daily or CX-5461/olaparib combination for 3 weeks. The PDX were harvested at a tumour volume of 700 mm 3 . Mean tumour volume (mm 3 ) (solid lines) ±95% CI (shaded region) and tumour volume of all individual mice (hashed lines) and corresponding Kaplan-Meier survival analysis. Censored events are represented by crosses on Kaplan-Meier plot. n indicates individual mice. c Schematic of CIdU and IdU pulse-labelling (top). OVCAR8 RAD51C KO cells or d WEHICS62 cell line derived from PDX#62 29 were sequentially labelled and either processed or treated with 2 mM hydroxyurea (HU) ± 1 μM CX-5461 for 3 h . Fibres were processed for DNA fibre analysis. n = 102 replication tracks of OVCAR8 RAD51C KO cells analyzed over two independent experiments, n = 236 replication tracks of WEHICS62 cells analysed over three independent experiments. Error bars represent mean ± SD. Statistical analysis in (C) was performed using a two-sided Mann–Whitney test and in ( d ) using two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). NS denotes non-significant p -value. e Co-IF analysis of pATR (T1989) and UBF in in WEHICS62 cells treated with vehicle or 100 nM CX-5461 for 24 h. Quantitation of signal intensity of the colocalized regions and total pATR was performed using CellProfiler. n = 506 cells per condition analysed over three independent experiments, error bars represent mean ± SD. Statistical analysis was performed using Mann–Whitney test. f BrdU cell cycle analysis of WEHCS62 cells treated with vehicle or 1 μM CX-5461 for 72 h (left panel), n = 3 biological replicates, mean ± SEM. Flow cytometry gating strategy is shown in Supplementary Fig. 3D . Statistical analysis was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). In vitro CX-5461 dose response proliferation time-course assessed using IncuCyte ZOOM. Representative of n = 3 biological replicates, mean ± SEM of five technical replicates.
Figure Legend Snippet: CX-5461 has significant therapeutic efficacy in HGSOC- patient-derived xenografts (PDX) models. a Responses observed in post-platinum treated BRCA2-mutant PDX#19 HGSOC-PDX and b PDX #62 with BRCA1 promoter methylation to CX-5461 and olaparib treatment in vivo. Recipient mice bearing the PDX were randomized to treatment with vehicle, 40 mg/kg CX-5461 twice a week, 50 mg/kg olaparib once daily or CX-5461/olaparib combination for 3 weeks. The PDX were harvested at a tumour volume of 700 mm 3 . Mean tumour volume (mm 3 ) (solid lines) ±95% CI (shaded region) and tumour volume of all individual mice (hashed lines) and corresponding Kaplan-Meier survival analysis. Censored events are represented by crosses on Kaplan-Meier plot. n indicates individual mice. c Schematic of CIdU and IdU pulse-labelling (top). OVCAR8 RAD51C KO cells or d WEHICS62 cell line derived from PDX#62 29 were sequentially labelled and either processed or treated with 2 mM hydroxyurea (HU) ± 1 μM CX-5461 for 3 h . Fibres were processed for DNA fibre analysis. n = 102 replication tracks of OVCAR8 RAD51C KO cells analyzed over two independent experiments, n = 236 replication tracks of WEHICS62 cells analysed over three independent experiments. Error bars represent mean ± SD. Statistical analysis in (C) was performed using a two-sided Mann–Whitney test and in ( d ) using two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). NS denotes non-significant p -value. e Co-IF analysis of pATR (T1989) and UBF in in WEHICS62 cells treated with vehicle or 100 nM CX-5461 for 24 h. Quantitation of signal intensity of the colocalized regions and total pATR was performed using CellProfiler. n = 506 cells per condition analysed over three independent experiments, error bars represent mean ± SD. Statistical analysis was performed using Mann–Whitney test. f BrdU cell cycle analysis of WEHCS62 cells treated with vehicle or 1 μM CX-5461 for 72 h (left panel), n = 3 biological replicates, mean ± SEM. Flow cytometry gating strategy is shown in Supplementary Fig. 3D . Statistical analysis was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). In vitro CX-5461 dose response proliferation time-course assessed using IncuCyte ZOOM. Representative of n = 3 biological replicates, mean ± SEM of five technical replicates.

Techniques Used: Derivative Assay, Mutagenesis, Methylation, In Vivo, Mouse Assay, MANN-WHITNEY, Quantitation Assay, Cell Cycle Assay, Flow Cytometry, In Vitro

CX-5461 cooperates with PARPi in inhibiting HGSOC cell growth. a Mini drug screen in OVCAR4 cells treated with increasing doses of cisplatin (0–1.11 μM), PARPi (BMN-673, 0–0.11 μM), ATMi (KU55933, 0–1.11 μM), ATRi (VE-821, 0–1.11 μM), ABT-119 (0–1.11 μM) or Everolimus (0-0.11 μM) ± 80 nM (GI 20 ) CX-5461. Colour-coding denotes the level of proliferation as measured by DAPI staining and imaging using Cellomics (green denotes reduced proliferation). Dose response of single drug treatments were corrected for vehicle control and the combination was corrected for response to 80 nM CX-5461, the average values of n = 5 are presented. The combination of CX-5461 with BMN-673 or ATRi is highlighted by yellow boxes. b Quantitation of SubG1 DNA content by PI staining of cells treated with vehicle, 100 nM CX-5461, 1 μM VE-821, 100 nM BMN-673 or in combination for 7 days ( n = 3 biologically independent experiments). Error bars represent mean ± SEM. Flow cytometry gating strategy is shown in Supplementary Fig. 3D. C ) Repr esentative BMN-673 dose response curves ± 30 nM CX-5461. Cell proliferation was measured using SRB assays at 5 days post-treatment. Dose response curves for BMN-673 were corrected for DMSO treatment control and the combination was corrected for response to 30 nM CX-5461. Representative of three biological replicates. Error bars represent mean ± SEM of n = 5 technical replicates. d BrdU cell cycle analysis of cells treated with vehicle, 1 μM CX-5461, 100 nM BMN-673 or in combination for 72 h as described in Fig. 3b and Supplementary Fig. 3D . n = 3, mean ± SEM. e In vitro proliferation time-course assessed by cell confluency using IncuCyte ZOOM. Representative of n = 3 biological replicates, mean ± SEM of five technical replicates. Dashed lines denote re-supplement of media with drugs. f CX-5461 and BMN-673 cooperate in inhibiting clonogenic survival. Representative image of n = 6 biologically independent experiments for OVCAR8 and OVCAR8 RAD51C KO cells and n = 3 biological replicates for FT282 cells, mean ± SEM. g Clonogenic assay of OV90 cells. n = 3 technical replicates. Statistical analysis (in b , d and f ) was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown).
Figure Legend Snippet: CX-5461 cooperates with PARPi in inhibiting HGSOC cell growth. a Mini drug screen in OVCAR4 cells treated with increasing doses of cisplatin (0–1.11 μM), PARPi (BMN-673, 0–0.11 μM), ATMi (KU55933, 0–1.11 μM), ATRi (VE-821, 0–1.11 μM), ABT-119 (0–1.11 μM) or Everolimus (0-0.11 μM) ± 80 nM (GI 20 ) CX-5461. Colour-coding denotes the level of proliferation as measured by DAPI staining and imaging using Cellomics (green denotes reduced proliferation). Dose response of single drug treatments were corrected for vehicle control and the combination was corrected for response to 80 nM CX-5461, the average values of n = 5 are presented. The combination of CX-5461 with BMN-673 or ATRi is highlighted by yellow boxes. b Quantitation of SubG1 DNA content by PI staining of cells treated with vehicle, 100 nM CX-5461, 1 μM VE-821, 100 nM BMN-673 or in combination for 7 days ( n = 3 biologically independent experiments). Error bars represent mean ± SEM. Flow cytometry gating strategy is shown in Supplementary Fig. 3D. C ) Repr esentative BMN-673 dose response curves ± 30 nM CX-5461. Cell proliferation was measured using SRB assays at 5 days post-treatment. Dose response curves for BMN-673 were corrected for DMSO treatment control and the combination was corrected for response to 30 nM CX-5461. Representative of three biological replicates. Error bars represent mean ± SEM of n = 5 technical replicates. d BrdU cell cycle analysis of cells treated with vehicle, 1 μM CX-5461, 100 nM BMN-673 or in combination for 72 h as described in Fig. 3b and Supplementary Fig. 3D . n = 3, mean ± SEM. e In vitro proliferation time-course assessed by cell confluency using IncuCyte ZOOM. Representative of n = 3 biological replicates, mean ± SEM of five technical replicates. Dashed lines denote re-supplement of media with drugs. f CX-5461 and BMN-673 cooperate in inhibiting clonogenic survival. Representative image of n = 6 biologically independent experiments for OVCAR8 and OVCAR8 RAD51C KO cells and n = 3 biological replicates for FT282 cells, mean ± SEM. g Clonogenic assay of OV90 cells. n = 3 technical replicates. Statistical analysis (in b , d and f ) was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown).

Techniques Used: Staining, Imaging, Quantitation Assay, Flow Cytometry, Sulforhodamine B Assay, Cell Cycle Assay, In Vitro, Clonogenic Assay

CX-5461 is synthetic lethal with HRD in HGSOC. a In vitro CX-5461 dose response proliferation time course, assessed by cell confluency using IncuCyte ZOOM of OVCAR8 and OVCAR8 RAD51C KO cell lines. Representative of two biologically independent experiments. Error bars represent mean ± SEM of n = 5 technical replicates. b Cell cycle analysis of cells treated with vehicle, 100 nM or 1 μM CX-5461 for 48 hours and 72 h and labelled with BrdU for 30 min prior to harvest. Analytical FACS analysis of BrdU incorporation as a function of DNA content was performed (Representative plots and gating strategy are shown in Supplementary Fig. 3D ). Histogram plots displaying the percentage of G0/G1 (blue) and G2M (green) and S-phase BrdU-labelled (red) cell populations. Error bars represent mean ± SEM of n = 3 biologically independent experiments. c Quantitation of cell cycle profiles using FUCCI-labelled cells treated with vehicle, 100 nM or 1 μM CX-5461 for 48 and 72 h. Representative flow cytometry profiles and gating strategy are shown in Supplementary Fig. 3E . Error bars represent mean ± SEM of n = 3 biologically independent experiments. d Histogram plots displaying the percentage of cells with > 4n DNA content (top panel) and Sub G0/G1 cell populations (bottom panel) as detected by BrdU/PI cell cycle analysis described in ( b ) and Supplementary Fig. 3D , ( n = 3 biologically independent experiments, n = 7 for OVCAR8 and OVCA8 RAD51C KO cells treated with vehicle or 1 μM CX-5461 for 72 h), error bars represent mean ± SEM. Statistical analysis in B-D was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). e Western blot analysis of cells treated with either vehicle, 100 nM or 1 μM CX-5461 for 6 and 24 h. Representative of n = 3 biologically independent experiments. Blots shown are of samples derived from the same experiment and were processed in parallel. Loading controls Vinculin and Actin were processed by re-probing the blots. Full sized scan of western blots are provided in Supplementary Fig. 10 .
Figure Legend Snippet: CX-5461 is synthetic lethal with HRD in HGSOC. a In vitro CX-5461 dose response proliferation time course, assessed by cell confluency using IncuCyte ZOOM of OVCAR8 and OVCAR8 RAD51C KO cell lines. Representative of two biologically independent experiments. Error bars represent mean ± SEM of n = 5 technical replicates. b Cell cycle analysis of cells treated with vehicle, 100 nM or 1 μM CX-5461 for 48 hours and 72 h and labelled with BrdU for 30 min prior to harvest. Analytical FACS analysis of BrdU incorporation as a function of DNA content was performed (Representative plots and gating strategy are shown in Supplementary Fig. 3D ). Histogram plots displaying the percentage of G0/G1 (blue) and G2M (green) and S-phase BrdU-labelled (red) cell populations. Error bars represent mean ± SEM of n = 3 biologically independent experiments. c Quantitation of cell cycle profiles using FUCCI-labelled cells treated with vehicle, 100 nM or 1 μM CX-5461 for 48 and 72 h. Representative flow cytometry profiles and gating strategy are shown in Supplementary Fig. 3E . Error bars represent mean ± SEM of n = 3 biologically independent experiments. d Histogram plots displaying the percentage of cells with > 4n DNA content (top panel) and Sub G0/G1 cell populations (bottom panel) as detected by BrdU/PI cell cycle analysis described in ( b ) and Supplementary Fig. 3D , ( n = 3 biologically independent experiments, n = 7 for OVCAR8 and OVCA8 RAD51C KO cells treated with vehicle or 1 μM CX-5461 for 72 h), error bars represent mean ± SEM. Statistical analysis in B-D was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p -values are shown). e Western blot analysis of cells treated with either vehicle, 100 nM or 1 μM CX-5461 for 6 and 24 h. Representative of n = 3 biologically independent experiments. Blots shown are of samples derived from the same experiment and were processed in parallel. Loading controls Vinculin and Actin were processed by re-probing the blots. Full sized scan of western blots are provided in Supplementary Fig. 10 .

Techniques Used: In Vitro, Cell Cycle Assay, FACS, BrdU Incorporation Assay, Quantitation Assay, Flow Cytometry, Western Blot, Derivative Assay

22) Product Images from "Combination therapy with dendritic cell vaccine and programmed death ligand 1 immune checkpoint inhibitor for hepatocellular carcinoma in an orthotopic mouse model"

Article Title: Combination therapy with dendritic cell vaccine and programmed death ligand 1 immune checkpoint inhibitor for hepatocellular carcinoma in an orthotopic mouse model

Journal: Therapeutic Advances in Medical Oncology

doi: 10.1177/1758835920922034

Functional characterization of the mDC. (a) For antigen uptake assay, the iDC and mDC were either remained untreated (black solid curves) or incubated with FITC-dextran at 37°C (red solid curves) or on ice (cyan solid curves), followed by flow cytometry analysis. The dextran-treated cells whose FITC intensity at 37°C was higher than that on ice were gated and considered as the cells with the capacity to uptake dextran. Shown are the representative results of three independent experiments. (b) The frequency of the cells positive for FITC-dextran was calculated as the percentage of all analyzed cells. Data represents the mean with SEM error bars of three independent experiments. ** p
Figure Legend Snippet: Functional characterization of the mDC. (a) For antigen uptake assay, the iDC and mDC were either remained untreated (black solid curves) or incubated with FITC-dextran at 37°C (red solid curves) or on ice (cyan solid curves), followed by flow cytometry analysis. The dextran-treated cells whose FITC intensity at 37°C was higher than that on ice were gated and considered as the cells with the capacity to uptake dextran. Shown are the representative results of three independent experiments. (b) The frequency of the cells positive for FITC-dextran was calculated as the percentage of all analyzed cells. Data represents the mean with SEM error bars of three independent experiments. ** p

Techniques Used: Functional Assay, Incubation, Flow Cytometry

Generation and morphological and phenotypical characterization of the mDC. (a) A schematic diagram illustrating the generation of the mDC from mouse bone marrow. The iDC expressed high levels of CD11c but low levels of CD40, CD80, and CD86 compared with the mDC expressing high levels of these four molecules. (b) Cell morphology examined by inverted phase-contrast microscopy. Black arrows indicate the dendritic protrusions of the suspended cells. Scale bar is shown in the bottom right corner of each image. Original magnification, ×20 (Day 1 and Day 3); ×40 (Day 6 and Day 7). (c) Representative result of flow cytometry analysis of the expression of the DC surface markers, including CD11c, CD40, CD80, and CD86 on the iDC and mDC. For the detection of each marker, the iDC and mDC were either stained with antibodies of each marker (orange and red solid curves, respectively) or isotype-matched control antibodies (cyan solid curves) or remained unstained (black solid curves). The stained cells whose FITC intensity was higher than that of the cells stained with isotype-matched control antibodies were considered as the cells positive for the indicated markers. The number of the cells expressing the indicated markers was calculated as the percentage of all analyzed cells and is shown in the upper right corner of each graph. DC, dendritic cells; FITC, fluorescein isothiocyanate; HCC, hepatocellular carcinoma; iDC, immature DC; LPS, lipopolysaccharide; mDC, mature DC.
Figure Legend Snippet: Generation and morphological and phenotypical characterization of the mDC. (a) A schematic diagram illustrating the generation of the mDC from mouse bone marrow. The iDC expressed high levels of CD11c but low levels of CD40, CD80, and CD86 compared with the mDC expressing high levels of these four molecules. (b) Cell morphology examined by inverted phase-contrast microscopy. Black arrows indicate the dendritic protrusions of the suspended cells. Scale bar is shown in the bottom right corner of each image. Original magnification, ×20 (Day 1 and Day 3); ×40 (Day 6 and Day 7). (c) Representative result of flow cytometry analysis of the expression of the DC surface markers, including CD11c, CD40, CD80, and CD86 on the iDC and mDC. For the detection of each marker, the iDC and mDC were either stained with antibodies of each marker (orange and red solid curves, respectively) or isotype-matched control antibodies (cyan solid curves) or remained unstained (black solid curves). The stained cells whose FITC intensity was higher than that of the cells stained with isotype-matched control antibodies were considered as the cells positive for the indicated markers. The number of the cells expressing the indicated markers was calculated as the percentage of all analyzed cells and is shown in the upper right corner of each graph. DC, dendritic cells; FITC, fluorescein isothiocyanate; HCC, hepatocellular carcinoma; iDC, immature DC; LPS, lipopolysaccharide; mDC, mature DC.

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

23) Product Images from "Interspecies differences in proteome turnover kinetics are correlated with lifespans and energetic demands"

Article Title: Interspecies differences in proteome turnover kinetics are correlated with lifespans and energetic demands

Journal: bioRxiv

doi: 10.1101/2020.04.25.061150

Differences in cellular ROS levels between mouse and naked mole rat cells. A. Flow cytometry analysis of CellROX fluorescence (ROS levels) and forward scatter (cell size) for mouse (red) and naked mole rat (blue) cells. B. Geometric mean intensities of CellROX fluorescence in mouse (red) and naked mole rat (blue) cells. Biological replicates represent cell lines cultured from individual organisms. The two pairs of measurements for biological replicates 2 represent measurements of distinct growths of the same cell line. The two biological replicates were measured using different flow cytometers as described in Methods.
Figure Legend Snippet: Differences in cellular ROS levels between mouse and naked mole rat cells. A. Flow cytometry analysis of CellROX fluorescence (ROS levels) and forward scatter (cell size) for mouse (red) and naked mole rat (blue) cells. B. Geometric mean intensities of CellROX fluorescence in mouse (red) and naked mole rat (blue) cells. Biological replicates represent cell lines cultured from individual organisms. The two pairs of measurements for biological replicates 2 represent measurements of distinct growths of the same cell line. The two biological replicates were measured using different flow cytometers as described in Methods.

Techniques Used: Flow Cytometry, Fluorescence, Cell Culture

24) Product Images from "An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA"

Article Title: An Atelocollagen Coating for Efficient Local Gene Silencing by Using Small Interfering RNA

Journal: Molecular Therapy. Nucleic Acids

doi: 10.1016/j.omtn.2017.01.006

ICAM-1 Expression after Knockdown with ICAM-1 siRNA with or without Lipofectamine 2000 Mediated by 0.008% and 0.032% ATCOL Transfection Coatings Glass slides were coated with ATCOL transfection solution and put into a 24-well plate. 75,000 EA.hy926 cells were cultivated on coated glass slides for 48 hr and were then activated with TNF-α. ICAM-1 expression was analyzed by flow cytometry. (A) Coatings consisting of 0.008% ATCOL and 1, 2.5, or 5 μg siICAM-1 with or without Lipofectamine 2000. 0.008% ATCOL coatings with the same amounts of siSCR (scrambled siRNA) and Lipofectamine 2000 served as a control. (B) 0.032% ATCOL coatings with 1, 2.5, and 5 μg siICAM-1 and Lipofectamine 2000. siSCR served as a control. The control (+ TNF + Ab) was set to 100% in (A) and (B). Each bar in (A) and (B) represents the mean ± SEM (n = 3). **p
Figure Legend Snippet: ICAM-1 Expression after Knockdown with ICAM-1 siRNA with or without Lipofectamine 2000 Mediated by 0.008% and 0.032% ATCOL Transfection Coatings Glass slides were coated with ATCOL transfection solution and put into a 24-well plate. 75,000 EA.hy926 cells were cultivated on coated glass slides for 48 hr and were then activated with TNF-α. ICAM-1 expression was analyzed by flow cytometry. (A) Coatings consisting of 0.008% ATCOL and 1, 2.5, or 5 μg siICAM-1 with or without Lipofectamine 2000. 0.008% ATCOL coatings with the same amounts of siSCR (scrambled siRNA) and Lipofectamine 2000 served as a control. (B) 0.032% ATCOL coatings with 1, 2.5, and 5 μg siICAM-1 and Lipofectamine 2000. siSCR served as a control. The control (+ TNF + Ab) was set to 100% in (A) and (B). Each bar in (A) and (B) represents the mean ± SEM (n = 3). **p

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

ICAM-1 Expression after Long-Term Knockdown of ICAM-1 Examined by Flow Cytometry Glass slides were coated with ATCOL/siICAM-1 transfection solution for substrate-mediated transfection and laid in a 24-well plate. For conventional transfection in tissue culture, ATCOL/siICAM-1 transfection solution was added to the cell culture medium, where EA.hy926 cells were seeded 1 day prior. After each time point, cells were activated for 14 hr with TNF-α and analyzed by flow cytometry. ICAM-1 expression was calculated after setting the TNF-α control to 100%. Each bar in (A) and (B) represents the mean ± SEM (n = 3). *p
Figure Legend Snippet: ICAM-1 Expression after Long-Term Knockdown of ICAM-1 Examined by Flow Cytometry Glass slides were coated with ATCOL/siICAM-1 transfection solution for substrate-mediated transfection and laid in a 24-well plate. For conventional transfection in tissue culture, ATCOL/siICAM-1 transfection solution was added to the cell culture medium, where EA.hy926 cells were seeded 1 day prior. After each time point, cells were activated for 14 hr with TNF-α and analyzed by flow cytometry. ICAM-1 expression was calculated after setting the TNF-α control to 100%. Each bar in (A) and (B) represents the mean ± SEM (n = 3). *p

Techniques Used: Expressing, Flow Cytometry, Cytometry, Transfection, Cell Culture

Transfection Efficiency after ATCOL-Mediated Transfection of siRNA AF 488 with or without Lipofectamine 2000 (A) 75,000 EA.hy926 cells were cultivated on 0.008% ATCOL with incorporated siRNA complexed or noncomplexed with Lipofectamine 2000. Transfection efficiency was analyzed by flow cytometry after 48 hr of cultivation. (B) 75,000 EA.hy926 cells were cultivated on 0.032% ATCOL with siRNA complexed or noncomplexed with Lipofectamine 2000 and then treated as mentioned previously. Coatings without siRNA and Lipofectamine 2000 served as a control. Each bar represents the mean ± SEM (n = 3, or n = 6 for 0.032% ATCOL with Lipofectamine 2000). *p
Figure Legend Snippet: Transfection Efficiency after ATCOL-Mediated Transfection of siRNA AF 488 with or without Lipofectamine 2000 (A) 75,000 EA.hy926 cells were cultivated on 0.008% ATCOL with incorporated siRNA complexed or noncomplexed with Lipofectamine 2000. Transfection efficiency was analyzed by flow cytometry after 48 hr of cultivation. (B) 75,000 EA.hy926 cells were cultivated on 0.032% ATCOL with siRNA complexed or noncomplexed with Lipofectamine 2000 and then treated as mentioned previously. Coatings without siRNA and Lipofectamine 2000 served as a control. Each bar represents the mean ± SEM (n = 3, or n = 6 for 0.032% ATCOL with Lipofectamine 2000). *p

Techniques Used: Transfection, Flow Cytometry, Cytometry

25) Product Images from "Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects"

Article Title: Cluster Intradermal DNA Vaccination Rapidly Induces E7-specific CD8+ T Cell Immune Responses Leading to Therapeutic Antitumor Effects

Journal:

doi: 10.1038/gt.2008.53

Flow cytometry analysis of CD4 + Foxp3 + regulatory T cells and Gr-1 + CD11b + myeloid suppressor cells in peripheral blood of TC-1 tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine
Figure Legend Snippet: Flow cytometry analysis of CD4 + Foxp3 + regulatory T cells and Gr-1 + CD11b + myeloid suppressor cells in peripheral blood of TC-1 tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

Intracellular cytokine staining followed by flow cytometry analysis to characterize E7-specific CD8 + cell precursors in mice vaccinated with pNGVL4a-CRT/E7(detox)DNA vaccine before and after tumor resection
Figure Legend Snippet: Intracellular cytokine staining followed by flow cytometry analysis to characterize E7-specific CD8 + cell precursors in mice vaccinated with pNGVL4a-CRT/E7(detox)DNA vaccine before and after tumor resection

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

Intracellular cytokine staining followed by flow cytometry analysis to characterize the E7-specific CD8 + cell precursors in the tumor and draining lymph nodes of tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox)DNA vaccine
Figure Legend Snippet: Intracellular cytokine staining followed by flow cytometry analysis to characterize the E7-specific CD8 + cell precursors in the tumor and draining lymph nodes of tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox)DNA vaccine

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

Flow cytometry analysis of CD4 + Foxp3 + regulatory T cells and CD124 + CD11b + myeloid suppressor cells in tumor infiltrating lymphocytes of TC-1 tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine
Figure Legend Snippet: Flow cytometry analysis of CD4 + Foxp3 + regulatory T cells and CD124 + CD11b + myeloid suppressor cells in tumor infiltrating lymphocytes of TC-1 tumor bearing mice vaccinated with pNGVL4a-CRT/E7(detox) DNA vaccine

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay

26) Product Images from "Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function"

Article Title: Antibodies targeting human OX40 expand effector T cells and block inducible and natural regulatory T cell function

Journal: Journal of immunology (Baltimore, Md. : 1950)

doi: 10.4049/jimmunol.1202752

Identification of anti-hOX40 mAbs that inhibit induction of T cells. ( A ) Flow cytometry analysis of L cells and L cells expressing human OX40 (hOX40-L) mixed (1:1) in FACS buffer and incubated with 0.5 μg of FPLC-purified, anti-hOX40 mAb. Anti-hOX40
Figure Legend Snippet: Identification of anti-hOX40 mAbs that inhibit induction of T cells. ( A ) Flow cytometry analysis of L cells and L cells expressing human OX40 (hOX40-L) mixed (1:1) in FACS buffer and incubated with 0.5 μg of FPLC-purified, anti-hOX40 mAb. Anti-hOX40

Techniques Used: Flow Cytometry, Cytometry, Expressing, FACS, Incubation, Fast Protein Liquid Chromatography, Purification

27) Product Images from "A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells"

Article Title: A ligand-mediated nanovector for targeted gene delivery and transfection in cancer cells

Journal: Biomaterials

doi: 10.1016/j.biomaterials.2008.10.003

Cell fluorescence intensity analysis by flow cytometry. C6 cells were exposed to either CTX-modified or unmodified nanovectors at varying DNA concentrations.
Figure Legend Snippet: Cell fluorescence intensity analysis by flow cytometry. C6 cells were exposed to either CTX-modified or unmodified nanovectors at varying DNA concentrations.

Techniques Used: Fluorescence, Flow Cytometry, Cytometry, Modification

Transfection efficiencies of C6 and NIH3T3 cells by DNA complexed with different polymeric vectors, as determined by flow cytometry. The commercially available Lipofectamine was tested as a reference transfection reagent.
Figure Legend Snippet: Transfection efficiencies of C6 and NIH3T3 cells by DNA complexed with different polymeric vectors, as determined by flow cytometry. The commercially available Lipofectamine was tested as a reference transfection reagent.

Techniques Used: Transfection, Flow Cytometry, Cytometry

Analysis of transgene expression of GFP by C6 cells. Transfection efficiency is measured by flow cytometry after treatment with either P-PEG-AF-SIAX/DNA or P-PEG-AF-CTX/DNA at varying DNA concentrations.
Figure Legend Snippet: Analysis of transgene expression of GFP by C6 cells. Transfection efficiency is measured by flow cytometry after treatment with either P-PEG-AF-SIAX/DNA or P-PEG-AF-CTX/DNA at varying DNA concentrations.

Techniques Used: Expressing, Transfection, Flow Cytometry, Cytometry

Flow cytometry based analysis for relative uptake of nanovectors for targeted (C6) and non-targeted (NIH3T3) cells. C6 and NIH3T3 cells were exposed to either the targeting (P-PEG-AF-CTX/DNA) or non-targeting (P-PEG-AF-SIAX/DNA) nanovector at a DNA concentration
Figure Legend Snippet: Flow cytometry based analysis for relative uptake of nanovectors for targeted (C6) and non-targeted (NIH3T3) cells. C6 and NIH3T3 cells were exposed to either the targeting (P-PEG-AF-CTX/DNA) or non-targeting (P-PEG-AF-SIAX/DNA) nanovector at a DNA concentration

Techniques Used: Flow Cytometry, Cytometry, Concentration Assay

28) Product Images from "Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus"

Article Title: Expression of Interleukin-4 by Recombinant Respiratory Syncytial Virus Is Associated with Accelerated Inflammation and a Nonfunctional Cytotoxic T-Lymphocyte Response following Primary Infection but Not following Challenge with Wild-Type Virus

Journal:

doi: 10.1128/JVI.79.15.9515-9526.2005

Flow cytometry of pulmonary myeloid and lymphoid dendritic cells in mice infected with RSV/IL-4 or wt RSV. (A) Example of primary data from individual mice on day 4 following infection with the indicated virus. The population with high forward scatter
Figure Legend Snippet: Flow cytometry of pulmonary myeloid and lymphoid dendritic cells in mice infected with RSV/IL-4 or wt RSV. (A) Example of primary data from individual mice on day 4 following infection with the indicated virus. The population with high forward scatter

Techniques Used: Flow Cytometry, Cytometry, Mouse Assay, Infection

29) Product Images from "Tumor secreted ANGPTL2 facilitates recruitment of neutrophils to the lung to promote lung pre-metastatic niche formation and targeting ANGPTL2 signaling affects metastatic disease"

Article Title: Tumor secreted ANGPTL2 facilitates recruitment of neutrophils to the lung to promote lung pre-metastatic niche formation and targeting ANGPTL2 signaling affects metastatic disease

Journal: Oncotarget

doi: 10.18632/oncotarget.27433

Integrin α5β1 deficiency reduces neutrophil recruitment to the lung. ( A–B ) The proportions (A) and absolute numbers (B) of neutrophils in the lung were detected by flow cytometry in Itga5-floxed mice or WT littermates after with LM9 and K7M2 cells inoculation into tibia (Gating strategy for neutrophils is shown in Supplementary Figure 4). ( C ) Absolute numbers of neutrophils in the lungs were detected by flow cytometry in WT littermates after intratibial inoculation of LM9-shCtr, LM9-shANGPTL2, K7M2-shCtr and K7M2-shANGPTL2 cells at indicated weeks. ( D ) Serum was isolated from tumor bearing animals one week after intratibial injection of tumor cells and analyzed by ELISA according to the manufacturer’s instructions (R D Systems). Similar results were obtained in BALF (Supplementary Figure 6A–6B) ( E ) CXCL1, CXCL2, CXCL5, and CXCL12 transcripts were significantly lower in Itga5-floxed AT-II cells compared to WT AT-II cells. AT-II cells were isolated from tumor bearing animals after a week of intratibial injection with indicated cells. Subsequently, total RNA was extracted and used for the gene expression analysis of chemokines. Results were normalized to GAPDH. Unpaired Student’s t -tests, * p
Figure Legend Snippet: Integrin α5β1 deficiency reduces neutrophil recruitment to the lung. ( A–B ) The proportions (A) and absolute numbers (B) of neutrophils in the lung were detected by flow cytometry in Itga5-floxed mice or WT littermates after with LM9 and K7M2 cells inoculation into tibia (Gating strategy for neutrophils is shown in Supplementary Figure 4). ( C ) Absolute numbers of neutrophils in the lungs were detected by flow cytometry in WT littermates after intratibial inoculation of LM9-shCtr, LM9-shANGPTL2, K7M2-shCtr and K7M2-shANGPTL2 cells at indicated weeks. ( D ) Serum was isolated from tumor bearing animals one week after intratibial injection of tumor cells and analyzed by ELISA according to the manufacturer’s instructions (R D Systems). Similar results were obtained in BALF (Supplementary Figure 6A–6B) ( E ) CXCL1, CXCL2, CXCL5, and CXCL12 transcripts were significantly lower in Itga5-floxed AT-II cells compared to WT AT-II cells. AT-II cells were isolated from tumor bearing animals after a week of intratibial injection with indicated cells. Subsequently, total RNA was extracted and used for the gene expression analysis of chemokines. Results were normalized to GAPDH. Unpaired Student’s t -tests, * p

Techniques Used: Flow Cytometry, Mouse Assay, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Expressing

30) Product Images from "CircRNA_100367 regulated the radiation sensitivity of esophageal squamous cell carcinomas through miR-217/Wnt3 pathway"

Article Title: CircRNA_100367 regulated the radiation sensitivity of esophageal squamous cell carcinomas through miR-217/Wnt3 pathway

Journal: Aging (Albany NY)

doi: 10.18632/aging.102580

CircRNA_100367 exists in KYSE-150R cells with higher potency of endothelial to mesenchymal transformation (EMT). ( A ) The protein levels characteristic molecules (β-catenin, Vimentin, and Snail) of EMT in KYSE-150 and KYSE-150R cells were detected by western blot. ( B ) The migration of KYSE-150 and KYSE-150R cells were determined by transwell assay. ( C ) The CD133 positive cells of KYSE-150 and KYSE-150R cells were measured by flow cytometry. ( D ) Divergent primers and convergent primers were designed, and PCR product of cricRNA 100367 was detected in 1.5% agarose gel electrophoresis. ( E ) After the treatment of RNase R, circRNA_100367 and DCAF8 mRNA expressions in KYSE-150 and KYSE-150R cells were detected by qRT-PCR. *p
Figure Legend Snippet: CircRNA_100367 exists in KYSE-150R cells with higher potency of endothelial to mesenchymal transformation (EMT). ( A ) The protein levels characteristic molecules (β-catenin, Vimentin, and Snail) of EMT in KYSE-150 and KYSE-150R cells were detected by western blot. ( B ) The migration of KYSE-150 and KYSE-150R cells were determined by transwell assay. ( C ) The CD133 positive cells of KYSE-150 and KYSE-150R cells were measured by flow cytometry. ( D ) Divergent primers and convergent primers were designed, and PCR product of cricRNA 100367 was detected in 1.5% agarose gel electrophoresis. ( E ) After the treatment of RNase R, circRNA_100367 and DCAF8 mRNA expressions in KYSE-150 and KYSE-150R cells were detected by qRT-PCR. *p

Techniques Used: Transformation Assay, Western Blot, Migration, Transwell Assay, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Quantitative RT-PCR

31) Product Images from "β7 Integrin Inhibition Can Increase Intestinal Inflammation by Impairing Homing of CD25hiFoxP3+ Regulatory T Cells"

Article Title: β7 Integrin Inhibition Can Increase Intestinal Inflammation by Impairing Homing of CD25hiFoxP3+ Regulatory T Cells

Journal: Cellular and Molecular Gastroenterology and Hepatology

doi: 10.1016/j.jcmgh.2019.10.012

Validation of dual-label intensity homing assay. ( A ) GFP + Tregs were sorted with a FACSAria 2 (BD Biosciences) from Itgb7 +/+ Foxp3 GFP or Itgb7 -/- Foxp3 GFP mice and labeled with 1 or 10 μmol/L of eFluor670, respectively. Equal numbers (1 Χ 10 7 ) of differentially labeled Tregs were mixed and then injected intravenously into C57BL/6 recipient mice. Spleens were harvested 3 hours after injection and isolated cells were analyzed by flow cytometry. The representative histograms were gated on GFP + Tregs. As shown in this histogram, this differential labeling produces 2 readily distinguished cell populations. ( B ) The same experiment was performed as in panel A , with the cell types receiving each concentration of eFluor670 reversed, that is, Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice were labeled with 10 μmol/L or 1 μmol/L of eFluor670, respectively.
Figure Legend Snippet: Validation of dual-label intensity homing assay. ( A ) GFP + Tregs were sorted with a FACSAria 2 (BD Biosciences) from Itgb7 +/+ Foxp3 GFP or Itgb7 -/- Foxp3 GFP mice and labeled with 1 or 10 μmol/L of eFluor670, respectively. Equal numbers (1 Χ 10 7 ) of differentially labeled Tregs were mixed and then injected intravenously into C57BL/6 recipient mice. Spleens were harvested 3 hours after injection and isolated cells were analyzed by flow cytometry. The representative histograms were gated on GFP + Tregs. As shown in this histogram, this differential labeling produces 2 readily distinguished cell populations. ( B ) The same experiment was performed as in panel A , with the cell types receiving each concentration of eFluor670 reversed, that is, Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice were labeled with 10 μmol/L or 1 μmol/L of eFluor670, respectively.

Techniques Used: Mouse Assay, Labeling, Injection, Isolation, Flow Cytometry, Concentration Assay

CD25 and Foxp3 expression on Itgb7 -/- Tregs. Expression of CD25 and Foxp3 on Tregs from Itgb7 +/+ Foxp3 GFP or Itgb7 -/- Foxp3 GFP mice are shown. Splenocytes were stained with CD25 antibody and then analyzed by flow cytometry.
Figure Legend Snippet: CD25 and Foxp3 expression on Itgb7 -/- Tregs. Expression of CD25 and Foxp3 on Tregs from Itgb7 +/+ Foxp3 GFP or Itgb7 -/- Foxp3 GFP mice are shown. Splenocytes were stained with CD25 antibody and then analyzed by flow cytometry.

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

Treg markers and transcription factor expression on Itgb7 -/- Tregs. Expression of Treg markers CD103, CD152 (CTLA4), OX40, and KLRG1, as well as transcription factors T-bet and GATA3, are shown. Cells from ( A ) spleen and ( B ) colonic lamina propria were isolated, stained with the indicated antibody, and then analyzed by flow cytometry.
Figure Legend Snippet: Treg markers and transcription factor expression on Itgb7 -/- Tregs. Expression of Treg markers CD103, CD152 (CTLA4), OX40, and KLRG1, as well as transcription factors T-bet and GATA3, are shown. Cells from ( A ) spleen and ( B ) colonic lamina propria were isolated, stained with the indicated antibody, and then analyzed by flow cytometry.

Techniques Used: Expressing, Isolation, Staining, Flow Cytometry

β7-deficient Tregs show normal suppression in vitro. ( A and B ) Treg suppression function. Tregs isolated from CD45.2 congenic Itgb7 +/+ Foxp3 GFP (WT Treg) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- Treg) mice were mixed with responder cells at the indicated Treg/responder cell ratios. Responder cells are CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 - conventional T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL2. ( A ) CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry at 72 hours. ( B ) The proliferation index was determined by FlowJo software. ( C ) Intracellular expression of IL10 and TGFβ1 of GFP + Tregs from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Splenocytes were stimulated ex vivo with phorbol myristate acetate and ionomycin in the presence of monensin (for IL10) or brefeldin A (for TGF-β1) for 4 hours at 37°C. Cells were fixed and permeabilized before staining (n = 3). Data represent means ± SEM. Two-tailed t test. ( D ) In vivo competitive homing of Tregs to different lymphoid tissues. GFP + Tregs were sorted from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Lymphoid organs were isolated 3 hours after injection of Tregs before flow cytometry analysis. The ratio of Itgb7 -/- Tregs to Itgb7 +/+ Tregs ( Itgb7 -/- /WT) within various lymphoid tissues is shown ( n = 15). Data represent means ± SEM. One-way analysis of variance with the Bonferroni posttest. WT Tregs, Tregs from Itgb7 +/+ Foxp3 GFP mice; Itgb7 -/- Tregs, Tregs from Itgb7 -/- Foxp3 GFP mice. MFI, mean fluorescence intensity.
Figure Legend Snippet: β7-deficient Tregs show normal suppression in vitro. ( A and B ) Treg suppression function. Tregs isolated from CD45.2 congenic Itgb7 +/+ Foxp3 GFP (WT Treg) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- Treg) mice were mixed with responder cells at the indicated Treg/responder cell ratios. Responder cells are CFSE-labeled CD45.1 congenic C57BL/6 CD4 + CD25 - conventional T cells activated by anti-CD3 (5 μg/mL), anti-CD28 (5 μg/mL), and IL2. ( A ) CFSE populations gated on CD45.1 + cells were analyzed by flow cytometry at 72 hours. ( B ) The proliferation index was determined by FlowJo software. ( C ) Intracellular expression of IL10 and TGFβ1 of GFP + Tregs from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Splenocytes were stimulated ex vivo with phorbol myristate acetate and ionomycin in the presence of monensin (for IL10) or brefeldin A (for TGF-β1) for 4 hours at 37°C. Cells were fixed and permeabilized before staining (n = 3). Data represent means ± SEM. Two-tailed t test. ( D ) In vivo competitive homing of Tregs to different lymphoid tissues. GFP + Tregs were sorted from Itgb7 +/+ Foxp3 GFP (WT) or Itgb7 -/- Foxp3 GFP ( Itgb7 -/- ) mice. Lymphoid organs were isolated 3 hours after injection of Tregs before flow cytometry analysis. The ratio of Itgb7 -/- Tregs to Itgb7 +/+ Tregs ( Itgb7 -/- /WT) within various lymphoid tissues is shown ( n = 15). Data represent means ± SEM. One-way analysis of variance with the Bonferroni posttest. WT Tregs, Tregs from Itgb7 +/+ Foxp3 GFP mice; Itgb7 -/- Tregs, Tregs from Itgb7 -/- Foxp3 GFP mice. MFI, mean fluorescence intensity.

Techniques Used: In Vitro, Isolation, Mouse Assay, Labeling, Flow Cytometry, Software, Expressing, Ex Vivo, Staining, Two Tailed Test, In Vivo, Injection, Fluorescence

β7-deficient CD4 + T cells ameliorate adoptive T-cell–transfer–induced colitis. ( A and B ) CD4 + CD25 - CD45RB high Tconv cells (1 × 10 6 ) from Itgb7 +/+ or Itgb7 -/- mice were injected into Rag1 -/- mice. Changes in ( A ) body weight and ( B ) survival ratio are shown. Changes in body weight are shown as a percentage of the original weight. The number of mice in each group is indicated. Data represent means ± SEM. Two-way analysis of variance with the Bonferroni posttest. ( C ) In vivo competitive homing of CD4 + T cells to lymphoid tissues. CD4 + T cells were isolated from either Itgb7 +/+ or Itgb7 -/- mice, differentially labeled, and mixed before injection into C57BL/6 mice. CD4 + T cells homing to different lymphoid organs were analyzed by flow cytometry 3 hours after injection. The ratio of Itgb7 -/- CD4 + T cells to Itgb7 +/+ CD4 + T cells ( Itgb7 -/- /WT) from different lymphoid organs is shown ( n = 14). Data represent means ± SEM. One-way analysis of variance with the Bonferroni posttest. ** P
Figure Legend Snippet: β7-deficient CD4 + T cells ameliorate adoptive T-cell–transfer–induced colitis. ( A and B ) CD4 + CD25 - CD45RB high Tconv cells (1 × 10 6 ) from Itgb7 +/+ or Itgb7 -/- mice were injected into Rag1 -/- mice. Changes in ( A ) body weight and ( B ) survival ratio are shown. Changes in body weight are shown as a percentage of the original weight. The number of mice in each group is indicated. Data represent means ± SEM. Two-way analysis of variance with the Bonferroni posttest. ( C ) In vivo competitive homing of CD4 + T cells to lymphoid tissues. CD4 + T cells were isolated from either Itgb7 +/+ or Itgb7 -/- mice, differentially labeled, and mixed before injection into C57BL/6 mice. CD4 + T cells homing to different lymphoid organs were analyzed by flow cytometry 3 hours after injection. The ratio of Itgb7 -/- CD4 + T cells to Itgb7 +/+ CD4 + T cells ( Itgb7 -/- /WT) from different lymphoid organs is shown ( n = 14). Data represent means ± SEM. One-way analysis of variance with the Bonferroni posttest. ** P

Techniques Used: Mouse Assay, Injection, In Vivo, Isolation, Labeling, Flow Cytometry

32) Product Images from "Heme oxygenase‐1 deficiency triggers exhaustion of hematopoietic stem cells"

Article Title: Heme oxygenase‐1 deficiency triggers exhaustion of hematopoietic stem cells

Journal: EMBO Reports

doi: 10.15252/embr.201947895

HO‐1 −/− mice have disturbed frequency of ECs, CARs, and MQs in BM and altered production of hematopoietic cytokines HO‐1 −/− mice have more ECs and less CARs ( P = 0.053) in BM, while frequency of PαS does not differ. Young HO‐1 −/− mice have lower frequency of Sca‐1 high fraction among BM ECs, what resembles phenotype of BM ECs in old mice. Intracellular flow cytometry reveals (C) lower levels of SDF‐1α in HO‐1 −/− CARs and (D) lower levels of LAP protein in HO‐1 −/− ECs. Analysis of cell surface SCF revealed lower frequency of SCF + ECs in HO‐1 −/− while no differences among CARs. The frequency of (F) CD163 + MQs and (G) CD163 − MQs in BM of HO‐1 −/− mice is altered. Data information: Where individual data points not shown n = 4/group; unpaired two‐tailed t ‐test. * P
Figure Legend Snippet: HO‐1 −/− mice have disturbed frequency of ECs, CARs, and MQs in BM and altered production of hematopoietic cytokines HO‐1 −/− mice have more ECs and less CARs ( P = 0.053) in BM, while frequency of PαS does not differ. Young HO‐1 −/− mice have lower frequency of Sca‐1 high fraction among BM ECs, what resembles phenotype of BM ECs in old mice. Intracellular flow cytometry reveals (C) lower levels of SDF‐1α in HO‐1 −/− CARs and (D) lower levels of LAP protein in HO‐1 −/− ECs. Analysis of cell surface SCF revealed lower frequency of SCF + ECs in HO‐1 −/− while no differences among CARs. The frequency of (F) CD163 + MQs and (G) CD163 − MQs in BM of HO‐1 −/− mice is altered. Data information: Where individual data points not shown n = 4/group; unpaired two‐tailed t ‐test. * P

Techniques Used: Mouse Assay, Flow Cytometry, Two Tailed Test

HO‐1 is expressed in BM endothelial cells and pericytes Metaphysis region in the BM is rich in endomucin + capillaries expressing HO‐1. mp—metaphysis; gp—growth plate; scale bar 100 μm. The HO‐1‐positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm. HO‐1 is expressed by smaller endomucin + CD31 + capillaries (#) as well as in bigger endomucin −/low CD31 + arteries (*). CD31 − pericytes wrapping the artery also express HO‐1 (*); scale bar 20 μm. HO‐1‐positive capillaries in the metaphysis expressed CD31 and Sca‐1. The capillaries are enveloped by HO‐1‐expressing pericytes. Part of the HO‐1 + pericytes express Sca‐1 (#), while others show no or low Sca‐1 signal (*); scale bar 20 μm. Flow cytometry analysis revealed the highest expression of HO‐1 in CD31 + Sca‐1 + ECs. CAR and PαS populations also express HO‐1, while most of non‐hematopoietic CD45 − Ter119 − are HO‐1‐negative in steady‐state conditions. BM macrophages (MQs) express HO‐1. The MHCII high MQ expresses higher levels of HO‐1 than MHCII low MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO‐1 in comparison with MQ. HO‐1 expression on mRNA level quantified by (G) qPCR or (H) RNA‐seq. qPCR analysis based on two independent experiments n = 10–11/group, and RNA‐seq analysis has n = 3–4/group, two‐tailed t ‐test for two groups comparison, and one‐way ANOVA with Bonferroni post‐test for multiple group comparison. * P
Figure Legend Snippet: HO‐1 is expressed in BM endothelial cells and pericytes Metaphysis region in the BM is rich in endomucin + capillaries expressing HO‐1. mp—metaphysis; gp—growth plate; scale bar 100 μm. The HO‐1‐positive small capillaries in metaphysis express endomucin and CD31. Shown maximum intensity projection, scale bar 20 μm. HO‐1 is expressed by smaller endomucin + CD31 + capillaries (#) as well as in bigger endomucin −/low CD31 + arteries (*). CD31 − pericytes wrapping the artery also express HO‐1 (*); scale bar 20 μm. HO‐1‐positive capillaries in the metaphysis expressed CD31 and Sca‐1. The capillaries are enveloped by HO‐1‐expressing pericytes. Part of the HO‐1 + pericytes express Sca‐1 (#), while others show no or low Sca‐1 signal (*); scale bar 20 μm. Flow cytometry analysis revealed the highest expression of HO‐1 in CD31 + Sca‐1 + ECs. CAR and PαS populations also express HO‐1, while most of non‐hematopoietic CD45 − Ter119 − are HO‐1‐negative in steady‐state conditions. BM macrophages (MQs) express HO‐1. The MHCII high MQ expresses higher levels of HO‐1 than MHCII low MQ. Cells within whole HSPC compartment (LKS) express no or low levels of HO‐1 in comparison with MQ. HO‐1 expression on mRNA level quantified by (G) qPCR or (H) RNA‐seq. qPCR analysis based on two independent experiments n = 10–11/group, and RNA‐seq analysis has n = 3–4/group, two‐tailed t ‐test for two groups comparison, and one‐way ANOVA with Bonferroni post‐test for multiple group comparison. * P

Techniques Used: Expressing, Flow Cytometry, Real-time Polymerase Chain Reaction, RNA Sequencing Assay, Two Tailed Test

33) Product Images from "Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts"

Article Title: Low-dose radiation enhances therapeutic HPV DNA vaccination in tumor-bearing hosts

Journal: Cancer immunology, immunotherapy : CII

doi: 10.1007/s00262-008-0596-0

Flow cytometry analysis to demonstrate the expression of annexin V on radiotherapy treated TC-1 tumor cells
Figure Legend Snippet: Flow cytometry analysis to demonstrate the expression of annexin V on radiotherapy treated TC-1 tumor cells

Techniques Used: Flow Cytometry, Cytometry, Expressing

Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8 + T cells in tumor-bearing mice treated with radiotherapy and/or DNA vaccine
Figure Legend Snippet: Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8 + T cells in tumor-bearing mice treated with radiotherapy and/or DNA vaccine

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

Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8 + TILs in tumor-bearing mice treated with radiotherapy and/or DNA vaccine
Figure Legend Snippet: Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8 + TILs in tumor-bearing mice treated with radiotherapy and/or DNA vaccine

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

Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8 + T cells in the spleen and tumors of tumor-bearing mice treated with radiotherapy and/or DNA vaccine
Figure Legend Snippet: Intracellular cytokine staining followed by flow cytometry analysis to determine the number of E7-specific CD8 + T cells in the spleen and tumors of tumor-bearing mice treated with radiotherapy and/or DNA vaccine

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

34) Product Images from "Tumor Necrosis Factor Inhibits Mesenchymal Stem Cell Differentiation into Osteoblasts Via the Ubiquitin E3 Ligase Wwp1"

Article Title: Tumor Necrosis Factor Inhibits Mesenchymal Stem Cell Differentiation into Osteoblasts Via the Ubiquitin E3 Ligase Wwp1

Journal: Stem cells (Dayton, Ohio)

doi: 10.1002/stem.703

Characterization of a mesenchymal stem cell (MSC)-enriched population derived from bone marrow stromal cells (BMSCs) of adult mice. Wild-type BMSCs were cultured in α-minimal essential medium, passaged twice, and subjected to flow cytometry after
Figure Legend Snippet: Characterization of a mesenchymal stem cell (MSC)-enriched population derived from bone marrow stromal cells (BMSCs) of adult mice. Wild-type BMSCs were cultured in α-minimal essential medium, passaged twice, and subjected to flow cytometry after

Techniques Used: Derivative Assay, Mouse Assay, Cell Culture, Flow Cytometry, Cytometry

35) Product Images from "A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation"

Article Title: A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.2335201100

( A ) GalNAc and acetylated azido GalNAc analogs: Ac 4 GalNAz, Ac 4 2AzGal, and Ac 4 6AzGalNAz. ( B ) Evaluation of acetylated azido GalNAc analogs (50 μM) for metabolic incorporation into CHO cell surface glycoproteins. Cells were incubated with the compounds, stained with phosphine-FLAG followed by a FITC-α-FLAG antibody, and analyzed by flow cytometry. ( C ) Dose-dependent incorporation of GalNAz into CHO cell surface glycoproteins. Cells were analyzed for cell surface azides as in B . ( D ) Competitive metabolism of 50 μM Ac 4 GalNAz and various concentrations of Gal, GlcNAc, or GalNAc. Cells were analyzed for cell surface azides as in B . MFI, mean fluorescence intensity in arbitrary units. Data points represent the average of duplicate experiments. Bars indicate high and low values.
Figure Legend Snippet: ( A ) GalNAc and acetylated azido GalNAc analogs: Ac 4 GalNAz, Ac 4 2AzGal, and Ac 4 6AzGalNAz. ( B ) Evaluation of acetylated azido GalNAc analogs (50 μM) for metabolic incorporation into CHO cell surface glycoproteins. Cells were incubated with the compounds, stained with phosphine-FLAG followed by a FITC-α-FLAG antibody, and analyzed by flow cytometry. ( C ) Dose-dependent incorporation of GalNAz into CHO cell surface glycoproteins. Cells were analyzed for cell surface azides as in B . ( D ) Competitive metabolism of 50 μM Ac 4 GalNAz and various concentrations of Gal, GlcNAc, or GalNAc. Cells were analyzed for cell surface azides as in B . MFI, mean fluorescence intensity in arbitrary units. Data points represent the average of duplicate experiments. Bars indicate high and low values.

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

Incorporation of GalNAz into the core positions of mucin-type O-linked glycoproteins on ldlD CHO cells. ( A ) Detection of Tn-antigen by FITC-HPA staining and flow cytometry analysis. ( B ) Detection of core 1 O-linked glycans by FITC-jacalin staining and flow cytometry analysis. MFI, mean fluorescence intensity in arbitrary units. Data points represent the average of duplicate experiments. Bars indicate high and low values.
Figure Legend Snippet: Incorporation of GalNAz into the core positions of mucin-type O-linked glycoproteins on ldlD CHO cells. ( A ) Detection of Tn-antigen by FITC-HPA staining and flow cytometry analysis. ( B ) Detection of core 1 O-linked glycans by FITC-jacalin staining and flow cytometry analysis. MFI, mean fluorescence intensity in arbitrary units. Data points represent the average of duplicate experiments. Bars indicate high and low values.

Techniques Used: Staining, Flow Cytometry, Cytometry, Fluorescence

Metabolic incorporation of GalNAz into glycoproteins of various mammalian cell lines. ( A ) Flow cytometry analysis of cell surface azides with (+) or without (–)50 μMAc 4 GalNAz. ( B ) α-FLAG Western blot analysis of total glycoprotein in cell lysates. ( C ) Coomassie staining of identical cell lysates indicating comparable levels of protein loading (≈50 μg per lane). COS-7, green monkey kidney cells; HeLa, human cervical epithelial tumor cells; NIH 3T3, human fibroblasts; Jurkat, human T cell lymphoma; MFI, mean fluorescence intensity in arbitrary units. Data points represent the average of duplicate experiments. Bars indicate high and low values.
Figure Legend Snippet: Metabolic incorporation of GalNAz into glycoproteins of various mammalian cell lines. ( A ) Flow cytometry analysis of cell surface azides with (+) or without (–)50 μMAc 4 GalNAz. ( B ) α-FLAG Western blot analysis of total glycoprotein in cell lysates. ( C ) Coomassie staining of identical cell lysates indicating comparable levels of protein loading (≈50 μg per lane). COS-7, green monkey kidney cells; HeLa, human cervical epithelial tumor cells; NIH 3T3, human fibroblasts; Jurkat, human T cell lymphoma; MFI, mean fluorescence intensity in arbitrary units. Data points represent the average of duplicate experiments. Bars indicate high and low values.

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

36) Product Images from "Off-Target Function of the Sonic-Hedgehog Inhibitor Cyclopamine in Mediating Apoptosis via Nitric Oxide-Dependent Neutral Sphingomyelinase 2/Ceramide Induction"

Article Title: Off-Target Function of the Sonic-Hedgehog Inhibitor Cyclopamine in Mediating Apoptosis via Nitric Oxide-Dependent Neutral Sphingomyelinase 2/Ceramide Induction

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-11-0705

Cyclopamine-mediated induction of nSMase2 is regulated selectively by NO generation in Daoy cells (A) Generation of NO/ONOO(−) in response to cyclopamine (10μg/mL for 4 hours) was measured by DAF, DCFDA, or DHE by flow cytometry in Daoy cells. Effects of L-NAME (50μmol/L; B) versus DETA (3mmol/L, 12 hours; C) on nSMase2 in the absence/presence of cyclopamine (10μg/mL, 24 hours) were measured by qPCR. Effects of siRNA-mediated knockdown of n-NOS on nSMase2 (D), or cell death in Daoy (E) and UM-SCC-14A (F) cells were measured by qPCR, or trypan blue exclusion assay, respectively. (G) Cells isolated from wt or fro/fro mice were treated with increasing concentrations of cyclopamine, and effects on cell growth were measured by trypan blue exclusion assay. Vehicle-treated cells were used as controls.
Figure Legend Snippet: Cyclopamine-mediated induction of nSMase2 is regulated selectively by NO generation in Daoy cells (A) Generation of NO/ONOO(−) in response to cyclopamine (10μg/mL for 4 hours) was measured by DAF, DCFDA, or DHE by flow cytometry in Daoy cells. Effects of L-NAME (50μmol/L; B) versus DETA (3mmol/L, 12 hours; C) on nSMase2 in the absence/presence of cyclopamine (10μg/mL, 24 hours) were measured by qPCR. Effects of siRNA-mediated knockdown of n-NOS on nSMase2 (D), or cell death in Daoy (E) and UM-SCC-14A (F) cells were measured by qPCR, or trypan blue exclusion assay, respectively. (G) Cells isolated from wt or fro/fro mice were treated with increasing concentrations of cyclopamine, and effects on cell growth were measured by trypan blue exclusion assay. Vehicle-treated cells were used as controls.

Techniques Used: Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Trypan Blue Exclusion Assay, Isolation, Mouse Assay

Cyclopamine-mediated increased nSMase2 is regulated by ROS/RNS generation Effects of oxidative stress via ROS/RNS generation on cyclopamine-mediated overexpression of nSMase2 were examined in the absence/presence of antioxidant NAC (500 μmol/L) using qPCR in Daoy (A) and UM-SCC-14A (B) cells. (C) ROS/RNS induction in response to cyclopamine (10μg/mL) at 0, 3, 6, 9, 12, and 15 hours was measured by DCFDA by flow cytometry in Daoy cells. (D) Effects of cyclopamine (10μg/ml, 4 hours) on ROS/RNS generation (left) and potential mitochondrial colocalization (middle and right) in Daoy cells were visualized by confocal microscopy using DCFDA (green) and mitotracker (red) as compared to vehicle-treated controls (lower and upper, respectively). (E) Effects of NAC (500μmol/L) on prevention of ROS/RNS generation in response to cyclopamine (10μg/mL, 6 hours) in UM-SCC-14A cells were measured using DCFDA by flow cytometry.
Figure Legend Snippet: Cyclopamine-mediated increased nSMase2 is regulated by ROS/RNS generation Effects of oxidative stress via ROS/RNS generation on cyclopamine-mediated overexpression of nSMase2 were examined in the absence/presence of antioxidant NAC (500 μmol/L) using qPCR in Daoy (A) and UM-SCC-14A (B) cells. (C) ROS/RNS induction in response to cyclopamine (10μg/mL) at 0, 3, 6, 9, 12, and 15 hours was measured by DCFDA by flow cytometry in Daoy cells. (D) Effects of cyclopamine (10μg/ml, 4 hours) on ROS/RNS generation (left) and potential mitochondrial colocalization (middle and right) in Daoy cells were visualized by confocal microscopy using DCFDA (green) and mitotracker (red) as compared to vehicle-treated controls (lower and upper, respectively). (E) Effects of NAC (500μmol/L) on prevention of ROS/RNS generation in response to cyclopamine (10μg/mL, 6 hours) in UM-SCC-14A cells were measured using DCFDA by flow cytometry.

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Confocal Microscopy

37) Product Images from "The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude mice"

Article Title: The hemopexin domain of MMP-9 inhibits angiogenesis and retards the growth of intracranial glioblastoma xenograft in nude mice

Journal: International journal of cancer. Journal international du cancer

doi: 10.1002/ijc.23951

Flow cytometry. ( a ) Effect of MMP-9-PEX on cell cycle distribution in HMECs. Cells were grown in vector alone (VC) or MMP-9-PEX SNB19 (PX1, PX2) cell conditioned medium for 24 hr and then the cells were harvested, stained with propidium iodide and cell
Figure Legend Snippet: Flow cytometry. ( a ) Effect of MMP-9-PEX on cell cycle distribution in HMECs. Cells were grown in vector alone (VC) or MMP-9-PEX SNB19 (PX1, PX2) cell conditioned medium for 24 hr and then the cells were harvested, stained with propidium iodide and cell

Techniques Used: Flow Cytometry, Cytometry, Plasmid Preparation, Staining

38) Product Images from "A Caspase-Dependent Pathway Is Involved in Wnt/?-Catenin Signaling Promoted Apoptosis in Bacillus Calmette-Guerin Infected RAW264.7 Macrophages"

Article Title: A Caspase-Dependent Pathway Is Involved in Wnt/?-Catenin Signaling Promoted Apoptosis in Bacillus Calmette-Guerin Infected RAW264.7 Macrophages

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms15035045

The activation of Wnt/β-catenin signaling promotes BCG-infected cell apoptosis. ( A ) An MTT assay determined the cell viability of RAW264.7 cells treated with indicated conditions for 24 h; ( B ) Representatives of dot plot from five independent experiments of flow cytometry analysis of the apoptotic fraction of RAW264.7 cells treated with indicated conditions for 24 h; ( C ) A time-dependent apoptotic cell death fraction of RAW264.7 cells treated with indicated condition for different time points. Compared to a naïve control, * p
Figure Legend Snippet: The activation of Wnt/β-catenin signaling promotes BCG-infected cell apoptosis. ( A ) An MTT assay determined the cell viability of RAW264.7 cells treated with indicated conditions for 24 h; ( B ) Representatives of dot plot from five independent experiments of flow cytometry analysis of the apoptotic fraction of RAW264.7 cells treated with indicated conditions for 24 h; ( C ) A time-dependent apoptotic cell death fraction of RAW264.7 cells treated with indicated condition for different time points. Compared to a naïve control, * p

Techniques Used: Activation Assay, Infection, MTT Assay, Flow Cytometry, Cytometry

Impact of Wnt/β-catenin signaling on mitochondrial membrane potential (ΔΨm) of RAW264.7 cells. ( A ) Representatives of dot plot of flow cytometry analysis for ΔΨm of cells treated with indicated condition for 12 h; ( B ) fractions of cells with low ΔΨm treated with indicated conditions for 12 h; ( C ) Immunoblots of apoptosis-related proteins in RAW264.7 cells treated with indicated conditions for 24 h. Compared to a naïve control, * p
Figure Legend Snippet: Impact of Wnt/β-catenin signaling on mitochondrial membrane potential (ΔΨm) of RAW264.7 cells. ( A ) Representatives of dot plot of flow cytometry analysis for ΔΨm of cells treated with indicated condition for 12 h; ( B ) fractions of cells with low ΔΨm treated with indicated conditions for 12 h; ( C ) Immunoblots of apoptosis-related proteins in RAW264.7 cells treated with indicated conditions for 24 h. Compared to a naïve control, * p

Techniques Used: Flow Cytometry, Cytometry, Western Blot

39) Product Images from "Antitumor effects of Xi Huang pills on MDA-MB-231 cells in vitro and in vivo"

Article Title: Antitumor effects of Xi Huang pills on MDA-MB-231 cells in vitro and in vivo

Journal: Molecular Medicine Reports

doi: 10.3892/mmr.2018.9203

Apoptosis of MDA-MB-231 cells treated with XHP. (A) Cell apoptosis as determined by flow cytometry analysis. Cells were treated with XHP (4, 8 and 12 mg/ml) for 24 h. Cells treated with medium (0 mg/ml XHP) were used as a control. The fluorescence intensity of Annexin V-FITC (x-axis) against the PI (y-axis) staining is shown. (B) XHP induced apoptosis in MDA-MB-231 cells in a dose-dependent manner. The apoptosis rate was defined as the early apoptosis rate plus the late apoptosis rate. The results represent the mean ± standard deviation (n=3). *P
Figure Legend Snippet: Apoptosis of MDA-MB-231 cells treated with XHP. (A) Cell apoptosis as determined by flow cytometry analysis. Cells were treated with XHP (4, 8 and 12 mg/ml) for 24 h. Cells treated with medium (0 mg/ml XHP) were used as a control. The fluorescence intensity of Annexin V-FITC (x-axis) against the PI (y-axis) staining is shown. (B) XHP induced apoptosis in MDA-MB-231 cells in a dose-dependent manner. The apoptosis rate was defined as the early apoptosis rate plus the late apoptosis rate. The results represent the mean ± standard deviation (n=3). *P

Techniques Used: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Fluorescence, Staining, Standard Deviation

Effect of XHP on the cell cycle distribution of MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with different concentrations of XHP (4, 8 and 12 mg/ml) for 48 h, and then harvested to determine cell cycle distribution by flow cytometry analysis. Cells treated with medium (0 mg/ml, XHP) were used as a control. (B) XHP induced cell cycle arrest at G 2 /M phase in a dose-dependent manner. Values represent the mean ± standard deviation (n=3). *P
Figure Legend Snippet: Effect of XHP on the cell cycle distribution of MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with different concentrations of XHP (4, 8 and 12 mg/ml) for 48 h, and then harvested to determine cell cycle distribution by flow cytometry analysis. Cells treated with medium (0 mg/ml, XHP) were used as a control. (B) XHP induced cell cycle arrest at G 2 /M phase in a dose-dependent manner. Values represent the mean ± standard deviation (n=3). *P

Techniques Used: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Standard Deviation

XHP induced Δψm depletion in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with different concentrations of XHP (4, 8 and 12 mg/ml) for 24 h and Δψm was analyzed by flow cytometry. Cells treated with medium (0 mg/ml XHP) were used as a control. The lower part of the circle shown the percentage of cell with lost Δψm after XHP treatment. (B) XHP induced the depletion of Δψm in a dose-dependent manner. Values represent the mean ± standard deviation (n=3). *P
Figure Legend Snippet: XHP induced Δψm depletion in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with different concentrations of XHP (4, 8 and 12 mg/ml) for 24 h and Δψm was analyzed by flow cytometry. Cells treated with medium (0 mg/ml XHP) were used as a control. The lower part of the circle shown the percentage of cell with lost Δψm after XHP treatment. (B) XHP induced the depletion of Δψm in a dose-dependent manner. Values represent the mean ± standard deviation (n=3). *P

Techniques Used: Multiple Displacement Amplification, Flow Cytometry, Cytometry, Standard Deviation

40) Product Images from "The origin and development of nonlymphoid tissue CD103+ DCs"

Article Title: The origin and development of nonlymphoid tissue CD103+ DCs

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20091756

Cytokine receptor expression profile among CD103 + and CD11b + tissue DC subsets. (A and B) DC subsets were sorted from the lung, liver, and spleen of C57BL/6 mice and RNA expression was measured by quantitative RT-PCR. Bar graphs present the relative expression of Flt3 (A) and MCSF-R (B) in lung and liver CD103 + (white) and CD11b + (black) DCs and spleen CD8 + (white) and CD8 − CD11b + (black) DCs. One representative experiment of three independent experiments is shown. (C and D) Dermal, lung, kidney, liver, and spleen cell suspensions were isolated from WT C57BL/6 and MCSF-R/EGFP mice and analyzed by flow cytometry. (C) Histograms show Flt3 cell surface expression (blue) or isotype antibody control (red) on gated CD103 + versus CD11b + DC subsets in the dermis, lung, kidney, and liver and CD8 + versus CD8 − CD11b + DCs in the spleen. (D) Histograms show MCSF-R/EGFP expression (blue) or WT C57BL/6 control (red) on gated DC subsets in the same tissue. These results are representative of three independent experiments.
Figure Legend Snippet: Cytokine receptor expression profile among CD103 + and CD11b + tissue DC subsets. (A and B) DC subsets were sorted from the lung, liver, and spleen of C57BL/6 mice and RNA expression was measured by quantitative RT-PCR. Bar graphs present the relative expression of Flt3 (A) and MCSF-R (B) in lung and liver CD103 + (white) and CD11b + (black) DCs and spleen CD8 + (white) and CD8 − CD11b + (black) DCs. One representative experiment of three independent experiments is shown. (C and D) Dermal, lung, kidney, liver, and spleen cell suspensions were isolated from WT C57BL/6 and MCSF-R/EGFP mice and analyzed by flow cytometry. (C) Histograms show Flt3 cell surface expression (blue) or isotype antibody control (red) on gated CD103 + versus CD11b + DC subsets in the dermis, lung, kidney, and liver and CD8 + versus CD8 − CD11b + DCs in the spleen. (D) Histograms show MCSF-R/EGFP expression (blue) or WT C57BL/6 control (red) on gated DC subsets in the same tissue. These results are representative of three independent experiments.

Techniques Used: Expressing, Mouse Assay, RNA Expression, Quantitative RT-PCR, Isolation, Flow Cytometry, Cytometry

The development of CD103 + DCs in tissues is also dependent on Id2 and IRF8. (A) Lung and liver CD103 + and CD11b + DC subsets and spleen CD8 + and CD8 − CD11b + DCs were isolated from C57BL/6 mice and mRNA expression of Id2 and IRF8 was measured by quantitative RT-PCR. Bar graphs present the relative expression of Id2 (left) and IRF8 (right). One representative experiment of two independent experiments is shown. (B and C) Nonlymphoid tissue DCs isolated from WT, Id2 KO, and IRF8 (BXH2) C57BL/6 mice were analyzed by flow cytometry. (B) Dot plots show CD103 and CD11b expression among gated DAPI − CD45 + CD11c + I-A + DCs in WT (left), Id2 KO mice (middle), and IRF8 (BXH2; right). (C) Bar graphs show the absolute numbers of CD103 + and CD11b + DC subsets among tissue DCs in WT (black), Id2 KO (white), and IRF8 (BXH2; gray) mice. Bars represent data from two pooled experiments ( n = 2–3). Error bars represent the mean ± SEM. (D) Lethally irradiated CD45.1 + recipient mice were reconstituted with 50% CD45.1 + WT and 50% CD45.2 + WT mixed BM or with 50% CD45.1 + WT and 50% CD45.2 + Id2 KO mixed BM and analyzed 2–3 mo after. Bar graphs show the relative contribution of Id2 KO CD45.2 + CD103 + DCs (white) among total CD103 + DCs and the relative contribution of Id2 KO CD45.2 + CD11b + DCs (black) among total CD11b + DCs. Spleen CD8 + (light gray) and CD11b + DC (black) subsets are also shown. Dark gray bars represent the percentage of Id2 KO CD45.2 + blood granulocytes chimerism. Data shown represent two pooled experiments ( n = 3). Error bars represent means ± SEM. P-values indicate the results of a Student's t test performed between the indicated groups.
Figure Legend Snippet: The development of CD103 + DCs in tissues is also dependent on Id2 and IRF8. (A) Lung and liver CD103 + and CD11b + DC subsets and spleen CD8 + and CD8 − CD11b + DCs were isolated from C57BL/6 mice and mRNA expression of Id2 and IRF8 was measured by quantitative RT-PCR. Bar graphs present the relative expression of Id2 (left) and IRF8 (right). One representative experiment of two independent experiments is shown. (B and C) Nonlymphoid tissue DCs isolated from WT, Id2 KO, and IRF8 (BXH2) C57BL/6 mice were analyzed by flow cytometry. (B) Dot plots show CD103 and CD11b expression among gated DAPI − CD45 + CD11c + I-A + DCs in WT (left), Id2 KO mice (middle), and IRF8 (BXH2; right). (C) Bar graphs show the absolute numbers of CD103 + and CD11b + DC subsets among tissue DCs in WT (black), Id2 KO (white), and IRF8 (BXH2; gray) mice. Bars represent data from two pooled experiments ( n = 2–3). Error bars represent the mean ± SEM. (D) Lethally irradiated CD45.1 + recipient mice were reconstituted with 50% CD45.1 + WT and 50% CD45.2 + WT mixed BM or with 50% CD45.1 + WT and 50% CD45.2 + Id2 KO mixed BM and analyzed 2–3 mo after. Bar graphs show the relative contribution of Id2 KO CD45.2 + CD103 + DCs (white) among total CD103 + DCs and the relative contribution of Id2 KO CD45.2 + CD11b + DCs (black) among total CD11b + DCs. Spleen CD8 + (light gray) and CD11b + DC (black) subsets are also shown. Dark gray bars represent the percentage of Id2 KO CD45.2 + blood granulocytes chimerism. Data shown represent two pooled experiments ( n = 3). Error bars represent means ± SEM. P-values indicate the results of a Student's t test performed between the indicated groups.

Techniques Used: Isolation, Mouse Assay, Expressing, Quantitative RT-PCR, Flow Cytometry, Cytometry, Irradiation

The development of tissue CD103 + DCs is dependent on Flt3 and Flt3L. (A–C) Nonlymphoid tissue DCs isolated from WT, Flt3 KO (A and B), and Flt3L KO (C) C57BL/6 mice were analyzed by flow cytometry. (A) Dot plots show CD103 and CD11b expression among gated DAPI − CD45 + CD11c + I-A + DCs in WT (left) and Flt3 KO mice (right). (B) Bar graphs show the absolute numbers of CD103 + and CD11b + DC subsets among tissue DCs in WT (black) versus Flt3 KO (white) versus Flt3L (gray) mice. Bars represent data from six pooled experiments ( n = 3–4). Error bars represent the mean ± SEM ( n = 3). (C) Lethally irradiated CD45.1 + recipient mice were reconstituted with 50% CD45.1 + WT and 50% CD45.2 + WT mixed BM or with 50% CD45.1 + WT and 50% CD45.2 + Flt3 KO mixed BM and analyzed 2–3 mo after. Bar graphs show the relative contribution of Flt3 KO CD45.2 + CD103 + DCs (white) among total CD103 + DCs and the relative contribution of Flt3 KO CD45.2 + CD11b + DCs (black) among total CD11b + DCs. Spleen CD8 + (light gray) and CD11b + DC (black) subsets are also shown. Dark gray bars represent the relative number of Flt3 KO CD45.2 + blood granulocyte chimerism. Data shown represent two pooled experiments ( n = 3). Error bars represent the means ± SEM. P-values indicate the results of a Student's t test performed between the indicated groups.
Figure Legend Snippet: The development of tissue CD103 + DCs is dependent on Flt3 and Flt3L. (A–C) Nonlymphoid tissue DCs isolated from WT, Flt3 KO (A and B), and Flt3L KO (C) C57BL/6 mice were analyzed by flow cytometry. (A) Dot plots show CD103 and CD11b expression among gated DAPI − CD45 + CD11c + I-A + DCs in WT (left) and Flt3 KO mice (right). (B) Bar graphs show the absolute numbers of CD103 + and CD11b + DC subsets among tissue DCs in WT (black) versus Flt3 KO (white) versus Flt3L (gray) mice. Bars represent data from six pooled experiments ( n = 3–4). Error bars represent the mean ± SEM ( n = 3). (C) Lethally irradiated CD45.1 + recipient mice were reconstituted with 50% CD45.1 + WT and 50% CD45.2 + WT mixed BM or with 50% CD45.1 + WT and 50% CD45.2 + Flt3 KO mixed BM and analyzed 2–3 mo after. Bar graphs show the relative contribution of Flt3 KO CD45.2 + CD103 + DCs (white) among total CD103 + DCs and the relative contribution of Flt3 KO CD45.2 + CD11b + DCs (black) among total CD11b + DCs. Spleen CD8 + (light gray) and CD11b + DC (black) subsets are also shown. Dark gray bars represent the relative number of Flt3 KO CD45.2 + blood granulocyte chimerism. Data shown represent two pooled experiments ( n = 3). Error bars represent the means ± SEM. P-values indicate the results of a Student's t test performed between the indicated groups.

Techniques Used: Isolation, Mouse Assay, Flow Cytometry, Cytometry, Expressing, Irradiation

Two major phenotypically distinct DC subsets coexist in the dermis, lung, liver, pancreatic islets, and kidney. (A and B) Dermal cell suspensions isolated from langerin/EGFP, CX3CR1/EGFP, and WT C57BL/6 mice were analyzed by flow cytometry. (A) Dot plot shows the expression of langerin and CD11b on gated DAPI − CD45 + CD11c + I-A + dermal DCs, allowing the discrimination of three DC subsets. (B) Histograms show the expression levels of CD103, EpCAM, CX3CR1, SIRP-α, and F4/80 on the gated population described in A. (C and D) Dermal, lung, liver, pancreatic islets, and kidney cell suspensions were isolated from langerin/EGFP, CX3CR1/EGFP, and WT C57BL/6 mice. (C) Dot plot shows the expression of CD103 versus CD11b (right) on gated nonlymphoid tissue DAPI − CD45 + CD11c + I-A + DCs (left). For dermal DCs analysis, DCs were also gated on EPCAM − to exclude LCs. (D) Histograms show overlays of cell surface marker expression on CD103 + (red) and CD11b + (blue) DC subsets. These results are representative of three independent experiments ( n = 2–3).
Figure Legend Snippet: Two major phenotypically distinct DC subsets coexist in the dermis, lung, liver, pancreatic islets, and kidney. (A and B) Dermal cell suspensions isolated from langerin/EGFP, CX3CR1/EGFP, and WT C57BL/6 mice were analyzed by flow cytometry. (A) Dot plot shows the expression of langerin and CD11b on gated DAPI − CD45 + CD11c + I-A + dermal DCs, allowing the discrimination of three DC subsets. (B) Histograms show the expression levels of CD103, EpCAM, CX3CR1, SIRP-α, and F4/80 on the gated population described in A. (C and D) Dermal, lung, liver, pancreatic islets, and kidney cell suspensions were isolated from langerin/EGFP, CX3CR1/EGFP, and WT C57BL/6 mice. (C) Dot plot shows the expression of CD103 versus CD11b (right) on gated nonlymphoid tissue DAPI − CD45 + CD11c + I-A + DCs (left). For dermal DCs analysis, DCs were also gated on EPCAM − to exclude LCs. (D) Histograms show overlays of cell surface marker expression on CD103 + (red) and CD11b + (blue) DC subsets. These results are representative of three independent experiments ( n = 2–3).

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

Turnover of nonlymphoid tissue DC subsets. (A and B) BrdU incorporation and proliferation index of tissue DC subsets were measured by flow cytometry. Dermal, lung, liver, kidney, and spleen cell suspensions were isolated from WT C57BL/6 mice. (A) Bar graphs show the relative numbers of BrdU + cells among CD103 + and CD11b + DC subsets in nonlymphoid tissues and among CD8 + and CD8 − CD11b + spleen DCs after a 2-h (white) or a 12-h (black) BrdU pulse. Bars represent data from two pooled experiments ( n = 3). Error bars represent the mean ± SEM. (B) Bar graph shows the percentage of proliferating cells (% S/G2/M) measured by DNA content among CD103 + (white) and CD11b + (black) DC subsets in nonlymphoid tissues and among CD8 + (grey) and CD8 − CD11b + (black) spleen DCs. Bars represent data from three pooled experiments ( n = 3). Error bars represent the mean ± SEM. (C) Tissue DC mixing in parabiotic mice. CD45.2 and CD45.1 C57BL/6 mice were surgically joined for 55 d before being separated. Graphs show the percentage of parabiont-derived DCs among each DC subset in the lung, liver, kidney, and spleen at different times after parabiont separation. Bars represent two separate parabionts at each time point. Error bars represent the mean ± SEM.
Figure Legend Snippet: Turnover of nonlymphoid tissue DC subsets. (A and B) BrdU incorporation and proliferation index of tissue DC subsets were measured by flow cytometry. Dermal, lung, liver, kidney, and spleen cell suspensions were isolated from WT C57BL/6 mice. (A) Bar graphs show the relative numbers of BrdU + cells among CD103 + and CD11b + DC subsets in nonlymphoid tissues and among CD8 + and CD8 − CD11b + spleen DCs after a 2-h (white) or a 12-h (black) BrdU pulse. Bars represent data from two pooled experiments ( n = 3). Error bars represent the mean ± SEM. (B) Bar graph shows the percentage of proliferating cells (% S/G2/M) measured by DNA content among CD103 + (white) and CD11b + (black) DC subsets in nonlymphoid tissues and among CD8 + (grey) and CD8 − CD11b + (black) spleen DCs. Bars represent data from three pooled experiments ( n = 3). Error bars represent the mean ± SEM. (C) Tissue DC mixing in parabiotic mice. CD45.2 and CD45.1 C57BL/6 mice were surgically joined for 55 d before being separated. Graphs show the percentage of parabiont-derived DCs among each DC subset in the lung, liver, kidney, and spleen at different times after parabiont separation. Bars represent two separate parabionts at each time point. Error bars represent the mean ± SEM.

Techniques Used: BrdU Incorporation Assay, Flow Cytometry, Cytometry, Isolation, Mouse Assay, Derivative Assay

Related Articles

Flow Cytometry:

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.

Article Title: Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice
Article Snippet: .. Flow cytometry analysis included a 4-color T-cell panel (CD8a-PE, CD4-APC, CD19-APC-Cy7and CD45r/B220-PerCP (BD Biosciences)) and a 6-color erythroid/myeloid panel (CD45-APC-Cy7, CD71-FITC, CD117-PE-Cy7, Ter119-APC, Ly-6G/Ly-6C (Gr1)-PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend). .. Cells were also stained with DAPI to determine cell viability.

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).

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity
Article Snippet: .. Mast cell phenotype was confirmed by flow cytometry analysis with α-c-kit (BD Biosciences) and α-FcεRIα (Biolegend, San Diego, CA) antibodies. ..

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) . ..

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.

Article Title: Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening
Article Snippet: .. The cells were then treated with various concentrations of doxycycline (Sigma-Aldrich) or IPTG (Sigma-Aldrich) for at least 5 days before flow cytometry analysis using an LSRFortessa X20 instrument (BD Biosciences). .. Expression of sgRNA by RT-qPCR Total RNA was first extracted with RNeasy mini kit (QIAGEN) and treated with DNase I (QIAGEN) following the manufacturer’s instructions.

Cytometry:

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.

Article Title: Insertional mutagenesis using the Sleeping Beauty transposon system identifies drivers of erythroleukemia in mice
Article Snippet: .. Flow cytometry analysis included a 4-color T-cell panel (CD8a-PE, CD4-APC, CD19-APC-Cy7and CD45r/B220-PerCP (BD Biosciences)) and a 6-color erythroid/myeloid panel (CD45-APC-Cy7, CD71-FITC, CD117-PE-Cy7, Ter119-APC, Ly-6G/Ly-6C (Gr1)-PerCP-Cy5.5, and B220-FITC (BD Biosciences) and CD3e Alexa Fluor 488 (BioLegend). .. Cells were also stained with DAPI to determine cell viability.

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).

Article Title: A Fusion Cytokine Coupling GMCSF to IL9 Induces Heterologous Receptor Clustering and STAT1 Hyperactivation through JAK2 Promiscuity
Article Snippet: .. Mast cell phenotype was confirmed by flow cytometry analysis with α-c-kit (BD Biosciences) and α-FcεRIα (Biolegend, San Diego, CA) antibodies. ..

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) . ..

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.

Article Title: Development of drug-inducible CRISPR-Cas9 systems for large-scale functional screening
Article Snippet: .. The cells were then treated with various concentrations of doxycycline (Sigma-Aldrich) or IPTG (Sigma-Aldrich) for at least 5 days before flow cytometry analysis using an LSRFortessa X20 instrument (BD Biosciences). .. Expression of sgRNA by RT-qPCR Total RNA was first extracted with RNeasy mini kit (QIAGEN) and treated with DNase I (QIAGEN) following the manufacturer’s instructions.

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
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) . ..

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
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    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
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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