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NR1H4 KO affects cell proliferation, survival, and apoptosis of HT29 colon cancer cells. (A) Cells (5 × 10 4 ) were seeded into 35 mm plates and incubated for 144 h. Cells were counted every 24 h and taken pictures at 10× magnification. (B) Cells (1 × 10 5 ) were plated into <t>6-well</t> plates and incubated for 72 h. The cell number was quantified. (C and D) Cells (200/well) were plated into 6-well plates and incubated for 10 days. Colonies were stained using the Diff-Quik system. Digital images were obtained by light microscopy (C) and quantified using ImageJ software (D). (E and F) Cells were grown for 48 h and harvested for flow cytometry to measure apoptotic cell death after staining with ANX V and PI. The percentage of non-apoptotic (PI+/ANX V–), early apoptotic (PI–/ANX V+), dying (PI+/ANX V+), and living (PI–/ANX V–) parental, MOCK, and NR1H4 KO HT29 cells were compared. (G) Cells were incubated for 72 h and harvested for immunoblotting. Some cells were grown for 24 h and exposed to Etoposide (30-100 µM) or DMSO (vehicle) for an additional 48 h, followed by immunoblotting. Results shown are representative of at least three independent experiments. Data are expressed as mean ± SE of at least three independent experiments. Statistical significance was assessed using unpaired Student’s t -test or one-way ANOVA. * P
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Images

1) Product Images from "The Role of Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4) in Colon Cancer Cell Survival through the Regulation of c-Myc Stability"

Article Title: The Role of Nuclear Receptor Subfamily 1 Group H Member 4 (NR1H4) in Colon Cancer Cell Survival through the Regulation of c-Myc Stability

Journal: Molecules and Cells

doi: 10.14348/molcells.2020.0041

NR1H4 KO affects cell proliferation, survival, and apoptosis of HT29 colon cancer cells. (A) Cells (5 × 10 4 ) were seeded into 35 mm plates and incubated for 144 h. Cells were counted every 24 h and taken pictures at 10× magnification. (B) Cells (1 × 10 5 ) were plated into 6-well plates and incubated for 72 h. The cell number was quantified. (C and D) Cells (200/well) were plated into 6-well plates and incubated for 10 days. Colonies were stained using the Diff-Quik system. Digital images were obtained by light microscopy (C) and quantified using ImageJ software (D). (E and F) Cells were grown for 48 h and harvested for flow cytometry to measure apoptotic cell death after staining with ANX V and PI. The percentage of non-apoptotic (PI+/ANX V–), early apoptotic (PI–/ANX V+), dying (PI+/ANX V+), and living (PI–/ANX V–) parental, MOCK, and NR1H4 KO HT29 cells were compared. (G) Cells were incubated for 72 h and harvested for immunoblotting. Some cells were grown for 24 h and exposed to Etoposide (30-100 µM) or DMSO (vehicle) for an additional 48 h, followed by immunoblotting. Results shown are representative of at least three independent experiments. Data are expressed as mean ± SE of at least three independent experiments. Statistical significance was assessed using unpaired Student’s t -test or one-way ANOVA. * P
Figure Legend Snippet: NR1H4 KO affects cell proliferation, survival, and apoptosis of HT29 colon cancer cells. (A) Cells (5 × 10 4 ) were seeded into 35 mm plates and incubated for 144 h. Cells were counted every 24 h and taken pictures at 10× magnification. (B) Cells (1 × 10 5 ) were plated into 6-well plates and incubated for 72 h. The cell number was quantified. (C and D) Cells (200/well) were plated into 6-well plates and incubated for 10 days. Colonies were stained using the Diff-Quik system. Digital images were obtained by light microscopy (C) and quantified using ImageJ software (D). (E and F) Cells were grown for 48 h and harvested for flow cytometry to measure apoptotic cell death after staining with ANX V and PI. The percentage of non-apoptotic (PI+/ANX V–), early apoptotic (PI–/ANX V+), dying (PI+/ANX V+), and living (PI–/ANX V–) parental, MOCK, and NR1H4 KO HT29 cells were compared. (G) Cells were incubated for 72 h and harvested for immunoblotting. Some cells were grown for 24 h and exposed to Etoposide (30-100 µM) or DMSO (vehicle) for an additional 48 h, followed by immunoblotting. Results shown are representative of at least three independent experiments. Data are expressed as mean ± SE of at least three independent experiments. Statistical significance was assessed using unpaired Student’s t -test or one-way ANOVA. * P

Techniques Used: Incubation, Staining, Diff-Quik, Light Microscopy, Software, Flow Cytometry

Generation of NR1H4 KO cell lines using CRISPR/CAS9 technology. (A) Simplified gene structure of NR1H4. (B) Workflow of the methodology used to generate NR1H4 KO cell lines. (C) Cells were grown for 24 h in 6-well plates and harvested for immunoblotting to evaluate NR1H4 expression. (D) Representative light microscopy images of sub-confluent cells. Results shown are representative of at least three independent experiments. Scale bars = 100 µm.
Figure Legend Snippet: Generation of NR1H4 KO cell lines using CRISPR/CAS9 technology. (A) Simplified gene structure of NR1H4. (B) Workflow of the methodology used to generate NR1H4 KO cell lines. (C) Cells were grown for 24 h in 6-well plates and harvested for immunoblotting to evaluate NR1H4 expression. (D) Representative light microscopy images of sub-confluent cells. Results shown are representative of at least three independent experiments. Scale bars = 100 µm.

Techniques Used: CRISPR, Expressing, Light Microscopy

NR1H4 activity is closely related to MYC expression and stability in HT29 colon cancer cells. (A) Cells were grown in 6-well plates for 24 h and transfected with siRNAs targeting NR1H4 for 48 h. (B) Cells were grown in 6-well plates for 24 h and exposed to 30 µM CDCA for the indicated period of time (0-60 min), following which cells were harvested for immunoblotting. Cells were grown in 6-well plates for 24 h and treated with 20 µM MG143 for 6 h, followed by immunoblotting. Cells were grown in 6-well plates for 24 h and harvested for immunoblotting. Results shown are representative of at least three independent experiments.
Figure Legend Snippet: NR1H4 activity is closely related to MYC expression and stability in HT29 colon cancer cells. (A) Cells were grown in 6-well plates for 24 h and transfected with siRNAs targeting NR1H4 for 48 h. (B) Cells were grown in 6-well plates for 24 h and exposed to 30 µM CDCA for the indicated period of time (0-60 min), following which cells were harvested for immunoblotting. Cells were grown in 6-well plates for 24 h and treated with 20 µM MG143 for 6 h, followed by immunoblotting. Cells were grown in 6-well plates for 24 h and harvested for immunoblotting. Results shown are representative of at least three independent experiments.

Techniques Used: Activity Assay, Expressing, Transfection

2) Product Images from "Safflor Yellow B Attenuates Ischemic Brain Injury via Downregulation of Long Noncoding AK046177 and Inhibition of MicroRNA-134 Expression in Rats"

Article Title: Safflor Yellow B Attenuates Ischemic Brain Injury via Downregulation of Long Noncoding AK046177 and Inhibition of MicroRNA-134 Expression in Rats

Journal: Oxidative Medicine and Cellular Longevity

doi: 10.1155/2020/4586839

Effect of safflor yellow B on TUNEL-positive cells, cell viability, and apoptosis. Rats were divided into six groups: sham, ischemia/reperfusion (I/R), AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir. (a) Representative images showing TUNEL-positive cells of cerebral cortex in different groups (×200 magnification; 1, 2, 3, 4, 5, and 6 represent sham, ischemia/reperfusion (I/R), AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir, respectively). (b) Apoptotic (TUNEL-positive) cells were detected, AI = (number of apoptotic cells/total cell number counted) × 100%( n = 3). Primary fetal cortical cells were seeded in 96-well and 6-well plates and divided into six groups: control, OGD/R, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir. Apart from the control group, all cells in the other groups were cultured in glucose-free DMEM and hypoxic conditions (1% O 2 /94% N 2 /5% CO 2 ) at 37°C for 4 h. Thereafter, all groups' media were replaced with normal DMEM, and culturing continued for 20 h of reoxygenation under normoxic conditions (95% air/5% CO 2 ). The cells were pretreated with SYB, AK046177, and miR-134 agomir before being exposed to OGD/R. Cell viability was detected by the MTT method. Cell apoptosis was analyzed using flow cytometry. (c1)–(c6) represent control, OGD/R, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir, respectively. (d) represents cell apoptosis ( n = 3). (e) represents cell viability ( n = 8). Data are presented as mean ± SD. One-way ANOVA test was used to determine statistical significance. ∗∗ P
Figure Legend Snippet: Effect of safflor yellow B on TUNEL-positive cells, cell viability, and apoptosis. Rats were divided into six groups: sham, ischemia/reperfusion (I/R), AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir. (a) Representative images showing TUNEL-positive cells of cerebral cortex in different groups (×200 magnification; 1, 2, 3, 4, 5, and 6 represent sham, ischemia/reperfusion (I/R), AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir, respectively). (b) Apoptotic (TUNEL-positive) cells were detected, AI = (number of apoptotic cells/total cell number counted) × 100%( n = 3). Primary fetal cortical cells were seeded in 96-well and 6-well plates and divided into six groups: control, OGD/R, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir. Apart from the control group, all cells in the other groups were cultured in glucose-free DMEM and hypoxic conditions (1% O 2 /94% N 2 /5% CO 2 ) at 37°C for 4 h. Thereafter, all groups' media were replaced with normal DMEM, and culturing continued for 20 h of reoxygenation under normoxic conditions (95% air/5% CO 2 ). The cells were pretreated with SYB, AK046177, and miR-134 agomir before being exposed to OGD/R. Cell viability was detected by the MTT method. Cell apoptosis was analyzed using flow cytometry. (c1)–(c6) represent control, OGD/R, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir, respectively. (d) represents cell apoptosis ( n = 3). (e) represents cell viability ( n = 8). Data are presented as mean ± SD. One-way ANOVA test was used to determine statistical significance. ∗∗ P

Techniques Used: TUNEL Assay, Cell Culture, MTT Assay, Flow Cytometry

Effect of safflor yellow B on mitochondrial structure and cell respiration. Primary fetal cortical cells were seeded in 96-well and 6-well plates and divided into six groups: control, OGD/R, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir. The cells were pretreated with the drugs including SYB, AK046177 siRNA, and miR-134 agomir before being dealing with OGD/R. (a1–a6) Mitochondrial structure was evaluated using a transmission electron microscope (×20,000 magnification, bar 1 μ m) and represent the mitochondria structure of the control group, the OGD/R group, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir, respectively. Transmission electron microscope shows normal mitochondrial structure (yellow solid line arrows), and pathological mitochondria with irregular shapes and swollen (red solid line arrow) and vesicular mitochondrial clusters (blue solid line arrow). Cellular oxygen consumption rate (OCR) was measured using an Oxygraph-2k system ( n = 3 experiments per condition). Data are presented as mean ± SD ( n = 3). One-way ANOVA test was used to determine statistical significance. ∗ P
Figure Legend Snippet: Effect of safflor yellow B on mitochondrial structure and cell respiration. Primary fetal cortical cells were seeded in 96-well and 6-well plates and divided into six groups: control, OGD/R, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir. The cells were pretreated with the drugs including SYB, AK046177 siRNA, and miR-134 agomir before being dealing with OGD/R. (a1–a6) Mitochondrial structure was evaluated using a transmission electron microscope (×20,000 magnification, bar 1 μ m) and represent the mitochondria structure of the control group, the OGD/R group, AK046177 siRNA, AK046177 siRNA+miR-134 agomir, SYB, and SYB+miR-134 agomir, respectively. Transmission electron microscope shows normal mitochondrial structure (yellow solid line arrows), and pathological mitochondria with irregular shapes and swollen (red solid line arrow) and vesicular mitochondrial clusters (blue solid line arrow). Cellular oxygen consumption rate (OCR) was measured using an Oxygraph-2k system ( n = 3 experiments per condition). Data are presented as mean ± SD ( n = 3). One-way ANOVA test was used to determine statistical significance. ∗ P

Techniques Used: Transmission Assay, Microscopy

3) Product Images from "Caudatin Isolated from Cynanchum auriculatum Inhibits Breast Cancer Stem Cell Formation via a GR/YAP Signaling"

Article Title: Caudatin Isolated from Cynanchum auriculatum Inhibits Breast Cancer Stem Cell Formation via a GR/YAP Signaling

Journal: Biomolecules

doi: 10.3390/biom10060925

The effects of caudatin on the proliferation and mammosphere formation of breast cancer cells. ( A ) MDA-MB-231 cells were cultured in a 96-well plate with caudatin for 24 h. MDA-MB-231 cell proliferation was assayed with an EZ-Cytox kit. ( B ) Caudatin and ciclesonide reduced mammosphere formation by breast cancer cells. Mammospheres derived from MDA-MB-231 cells were cultured in ultralow-attachment 6-well plates with CSC culture medium for seven days. The mammosphere formation efficiency (MFE) was examined with increasing concentrations of caudatin and ciclesonide. Images show representative mammospheres and were obtained by microscopy (scale bar: 100 μm). ( C ) Caudatin induced apoptosis in MDA-MB-231 cells. Apoptosis was determined using Annexin V/propidium iodide (PI) staining. ( D ) Apoptotic cells induced by caudatin were stained with Hoechst 33,342 dye (scale bar: 50 μm). ( E ) The migration of MDA-MB-231 cells with/without caudatin (Dulbecco’s Modified Eagle’s medium (DMEM)/0.5% fetal bovine serum (FBS)) was imaged at 0, 6 h and 12 h by a scratch assay (scale bar: 100 μm). The percent of inhibition in cell migration was expressed using untreated well at 100%. ( F ) The cell migration (without Matrigel) and invasion (with Matrigel) of MDA-MB-231 cells exposed to caudatin were determined by transwell assays (scale bar: 100 μm). ( G ) MDA-MB-231 cells were incubated in 6-well plates and treated with caudatin. Representative colony formation data were collected. The data from triplicate experiments are represented as the mean ± SD. ** p
Figure Legend Snippet: The effects of caudatin on the proliferation and mammosphere formation of breast cancer cells. ( A ) MDA-MB-231 cells were cultured in a 96-well plate with caudatin for 24 h. MDA-MB-231 cell proliferation was assayed with an EZ-Cytox kit. ( B ) Caudatin and ciclesonide reduced mammosphere formation by breast cancer cells. Mammospheres derived from MDA-MB-231 cells were cultured in ultralow-attachment 6-well plates with CSC culture medium for seven days. The mammosphere formation efficiency (MFE) was examined with increasing concentrations of caudatin and ciclesonide. Images show representative mammospheres and were obtained by microscopy (scale bar: 100 μm). ( C ) Caudatin induced apoptosis in MDA-MB-231 cells. Apoptosis was determined using Annexin V/propidium iodide (PI) staining. ( D ) Apoptotic cells induced by caudatin were stained with Hoechst 33,342 dye (scale bar: 50 μm). ( E ) The migration of MDA-MB-231 cells with/without caudatin (Dulbecco’s Modified Eagle’s medium (DMEM)/0.5% fetal bovine serum (FBS)) was imaged at 0, 6 h and 12 h by a scratch assay (scale bar: 100 μm). The percent of inhibition in cell migration was expressed using untreated well at 100%. ( F ) The cell migration (without Matrigel) and invasion (with Matrigel) of MDA-MB-231 cells exposed to caudatin were determined by transwell assays (scale bar: 100 μm). ( G ) MDA-MB-231 cells were incubated in 6-well plates and treated with caudatin. Representative colony formation data were collected. The data from triplicate experiments are represented as the mean ± SD. ** p

Techniques Used: Multiple Displacement Amplification, Cell Culture, Derivative Assay, Microscopy, Staining, Migration, Modification, Wound Healing Assay, Inhibition, Incubation

4) Product Images from "Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics"

Article Title: Evaluation of Fat Accumulation and Adipokine Production during the Long-Term Adipogenic Differentiation of Porcine Intramuscular Preadipocytes and Study of the Influence of Immunobiotics

Journal: Cells

doi: 10.3390/cells9071715

Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of 6-well plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p
Figure Legend Snippet: Immunobiotic mediated regulation of adipocyte differentiation in an immune cell-adipocyte co-culture model. The mRNA expressions of PPARγ and GLUT4 resulted from various immunobiotic lactic acid bacteria (LAB) stimulation into the co-culture model. A total of 1 × 10 6 immune cells/well of 6-well plate were cultured on adipocytes 4-days differentiated. LAB samples (5 × 10 8 cells/well) also added into the adipocyte plate and maintained for further 2 or 4 Days. All data shown are the average ±SD of 3 independent experiments performed in triplicate. The asterisk (*) indicated statistical differences when compared with control at the significance level of p

Techniques Used: Co-Culture Assay, Cell Culture

Immunobiotic mediated inflammatory responses in the antigen presenting cells (APCs) of Payer’s patches. The APCs were seeded at 1 × 10 6 cells/well of 6-well plate and cultured with each bacterial strain (5 × 10 8 cells/well) separately for 24 h and the mRNA expressions of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-β and IFN-γ) were analyzed with RT-qPCR. Data shown are the mean ±SD of 3 independent experiments performed in triplicates. The asterisks: (*) and (**) indicated statistical differences with significant levels of p
Figure Legend Snippet: Immunobiotic mediated inflammatory responses in the antigen presenting cells (APCs) of Payer’s patches. The APCs were seeded at 1 × 10 6 cells/well of 6-well plate and cultured with each bacterial strain (5 × 10 8 cells/well) separately for 24 h and the mRNA expressions of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, IFN-β and IFN-γ) were analyzed with RT-qPCR. Data shown are the mean ±SD of 3 independent experiments performed in triplicates. The asterisks: (*) and (**) indicated statistical differences with significant levels of p

Techniques Used: Cell Culture, Quantitative RT-PCR

Immunobiotic mediated regulation of fat accumulation and inflammatory responses in an immune cell-adipocyte co-culture model. A total of 1 × 10 6 immune cells/well of a 6-well plate were cultured on adipocytes 4-days differentiated. Bacterial samples (5 × 10 8 cells/well) were also added and cultured for 2 or 4 days. Adipocyte cells were stained with Oil red O stained and fat accumulation in the adipocytes was quantitatively analyzed by image J software. The ELISA-based concentration of CCL2 protein in supernatant of adipocyte-culture stimulated by immune cells and lactic acid bacteria (LAB). All data shown are the mean ± SD of three independent experiments performed in triplicate. The asterisks: ‘*’ and ‘**’ indicated statistical differences either between bars of panel-a and control or between bars of panel-b and IMU, with significant levels of p
Figure Legend Snippet: Immunobiotic mediated regulation of fat accumulation and inflammatory responses in an immune cell-adipocyte co-culture model. A total of 1 × 10 6 immune cells/well of a 6-well plate were cultured on adipocytes 4-days differentiated. Bacterial samples (5 × 10 8 cells/well) were also added and cultured for 2 or 4 days. Adipocyte cells were stained with Oil red O stained and fat accumulation in the adipocytes was quantitatively analyzed by image J software. The ELISA-based concentration of CCL2 protein in supernatant of adipocyte-culture stimulated by immune cells and lactic acid bacteria (LAB). All data shown are the mean ± SD of three independent experiments performed in triplicate. The asterisks: ‘*’ and ‘**’ indicated statistical differences either between bars of panel-a and control or between bars of panel-b and IMU, with significant levels of p

Techniques Used: Co-Culture Assay, Cell Culture, Staining, Software, Enzyme-linked Immunosorbent Assay, Concentration Assay

5) Product Images from "Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones"

Article Title: Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones

Journal: Cancers

doi: 10.3390/cancers11111690

Cytostatic and proapoptotic activity of the selected conjugates. ( A ) Colony formation assay. The 22Rv1 cells were treated with the indicated concentrations of the compounds for 48 h, seeded in 6-well plates and incubated for 14 days. Cancer cell colonies were stained and counted by naked eye. ( B ), Cell viability and IC 50 s estimated in 22Rv1 cells using the trypan blue exclusion assay after 48 h of treatment. ( C – E ), Flow cytometry analysis of 22Rv1 cells treated with the investigated compounds for 48 h. ( C , D ), PI single staining: analysis of cell cycle. Apoptotic cells were detected as sub-G1 population ( D ). ( E ), Annexin-V-FITC/PI double staining. Cells appeared in low right quadrant (Annexin-V-FITC + /PI - ) were considered to undergo early apoptosis. Flow cytometry data were analyzed and quantified using the Cell Quest Pro software. ( F , G ), The Western blotting analysis of the expression of pro- and anti-apoptotic proteins ( F ) as well as autophagy and AR-signaling related proteins ( G ) in 22Rv1 cells after 48 h of treatment. β-actin was used as a loading control. Cells treated with 10 µM of anisomycin (Aniso) for 48 h were used as a positive control. Statistical significance: * p
Figure Legend Snippet: Cytostatic and proapoptotic activity of the selected conjugates. ( A ) Colony formation assay. The 22Rv1 cells were treated with the indicated concentrations of the compounds for 48 h, seeded in 6-well plates and incubated for 14 days. Cancer cell colonies were stained and counted by naked eye. ( B ), Cell viability and IC 50 s estimated in 22Rv1 cells using the trypan blue exclusion assay after 48 h of treatment. ( C – E ), Flow cytometry analysis of 22Rv1 cells treated with the investigated compounds for 48 h. ( C , D ), PI single staining: analysis of cell cycle. Apoptotic cells were detected as sub-G1 population ( D ). ( E ), Annexin-V-FITC/PI double staining. Cells appeared in low right quadrant (Annexin-V-FITC + /PI - ) were considered to undergo early apoptosis. Flow cytometry data were analyzed and quantified using the Cell Quest Pro software. ( F , G ), The Western blotting analysis of the expression of pro- and anti-apoptotic proteins ( F ) as well as autophagy and AR-signaling related proteins ( G ) in 22Rv1 cells after 48 h of treatment. β-actin was used as a loading control. Cells treated with 10 µM of anisomycin (Aniso) for 48 h were used as a positive control. Statistical significance: * p

Techniques Used: Activity Assay, Colony Assay, Incubation, Staining, Trypan Blue Exclusion Assay, Flow Cytometry, Cytometry, Double Staining, Software, Western Blot, Expressing, Positive Control

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Multiple Displacement Amplification:

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Staining:

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Article Snippet: .. In brief, cells were pre-incubated overnight in 6-well plates (0.2 × 106 cells/well in 2 mL/well), treated with the drugs for 48 h. Then cells were harvested, fixed, stained with PI, and analyzed using FACS Calibur (BD Bioscience, San Jose, CA, USA) and BD Bioscience Cell Quest Pro v.5.2.1. software (BD Bioscience). .. Detection of Apoptotic Cells by Annexin-V-FITC/PI Double Staining Induction of apoptosis was analyzed by flow cytometry technique using annexin-V-FITC and propidium iodide (PI) double staining.

FACS:

Article Title: Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones
Article Snippet: .. In brief, cells were pre-incubated overnight in 6-well plates (0.2 × 106 cells/well in 2 mL/well), treated with the drugs for 48 h. Then cells were harvested, fixed, stained with PI, and analyzed using FACS Calibur (BD Bioscience, San Jose, CA, USA) and BD Bioscience Cell Quest Pro v.5.2.1. software (BD Bioscience). .. Detection of Apoptotic Cells by Annexin-V-FITC/PI Double Staining Induction of apoptosis was analyzed by flow cytometry technique using annexin-V-FITC and propidium iodide (PI) double staining.

Software:

Article Title: Successful Targeting of the Warburg Effect in Prostate Cancer by Glucose-Conjugated 1,4-Naphthoquinones
Article Snippet: .. In brief, cells were pre-incubated overnight in 6-well plates (0.2 × 106 cells/well in 2 mL/well), treated with the drugs for 48 h. Then cells were harvested, fixed, stained with PI, and analyzed using FACS Calibur (BD Bioscience, San Jose, CA, USA) and BD Bioscience Cell Quest Pro v.5.2.1. software (BD Bioscience). .. Detection of Apoptotic Cells by Annexin-V-FITC/PI Double Staining Induction of apoptosis was analyzed by flow cytometry technique using annexin-V-FITC and propidium iodide (PI) double staining.

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    Becton Dickinson dioscin
    ( A ) Effects of <t>dioscin</t> on cytochrome C release in Hela cells (800×, final magnification); ( B ) Effects of dioscin on cytochrome C release in SiHa cells (800×, final magnification).
    Dioscin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 91/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dioscin/product/Becton Dickinson
    Average 91 stars, based on 13 article reviews
    Price from $9.99 to $1999.99
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    93
    Becton Dickinson overexpression plasmids
    A) % Viability and B) % Cell proliferation upon <t>overexpression</t> of Robo 4 in PC3, DU145 and LNCaP cells; data from ≥3 independent experiments, *p
    Overexpression Plasmids, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/overexpression plasmids/product/Becton Dickinson
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    92
    Becton Dickinson bma097
    <t>BMA097</t> inhibits proliferation and induces apoptosis in breast cancer cells (A) Survival assay. Human breast cancer cells were treated with BMA097 at different concentrations for various times followed by MTT assay. (B–D) Apoptosis assay. Human breast cancer cells were treated with BMA097 at different concentrations (B and D) or at 2 μM for different times (C) followed by Annexin V/PI (B–C) or JC-1 staining (D) and FACS analysis of apoptosis and mitochondrial membrane potential (Δψ m ). (* p
    Bma097, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bma097/product/Becton Dickinson
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    88
    Becton Dickinson noncoated polyethylene terephthalate membranes
    SCH-479833 and SCH-527123 inhibit melanoma cell motility and invasion. Cells were plated on <t>noncoated</t> or Matrigel-coated membranes for motility ( A and B ) and invasion ( C and D ) assays and incubated for overnight. Serum-free medium containing 10 ng/mL CXCL-8 and SCH-479833 or SCH-527123 (10 ng/mL) or HPβCD was added to the lower chamber.The cells that did not migrate through the Matrigel and/or pores in the membrane were removed, and cells on the other side of the membrane were stained and photographed at ×200 magnification. Cells were counted in 10 random fields (×200) and expressed as the average number of cells per field of view. Number ± SE of migrated cells. Representative of three experiments done in triplicate. *, P
    Noncoated Polyethylene Terephthalate Membranes, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    ( A ) Effects of dioscin on cytochrome C release in Hela cells (800×, final magnification); ( B ) Effects of dioscin on cytochrome C release in SiHa cells (800×, final magnification).

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: ( A ) Effects of dioscin on cytochrome C release in Hela cells (800×, final magnification); ( B ) Effects of dioscin on cytochrome C release in SiHa cells (800×, final magnification).

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques:

    Morphological changes of HeLa cells treated by dioscin.

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Morphological changes of HeLa cells treated by dioscin.

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques:

    Dioscin induced DNA damage and cell cycle arrest of the HeLa and SaHa cells. ( A ) Dioscin induced DNA damage in HeLa cells by SCGE assay (200×, final magnification); ( B ) Dioscin induced DNA damage in SiHa cells by SCGE assay (200×, final magnification). Data are presented as mean ± SD ( n = 3). * p

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Dioscin induced DNA damage and cell cycle arrest of the HeLa and SaHa cells. ( A ) Dioscin induced DNA damage in HeLa cells by SCGE assay (200×, final magnification); ( B ) Dioscin induced DNA damage in SiHa cells by SCGE assay (200×, final magnification). Data are presented as mean ± SD ( n = 3). * p

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques: Single Cell Gel Electrophoresis

    The chemical structure of dioscin.

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: The chemical structure of dioscin.

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques:

    Effects of dioscin on ROS levels and Ca 2+ release. ( A ) ROS generation in HeLa cell treated by dioscin; ( B ) ROS generation in SiHa cell treated by dioscin; ( C ) Effects of dioscin on Ca 2+ level in HeLa cells by flow cytometry; ( D ) Effects of dioscin on Ca 2+ level in SiHa cells by flow cytometry. Data are presented as mean ± SD ( n = 5). * p

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Effects of dioscin on ROS levels and Ca 2+ release. ( A ) ROS generation in HeLa cell treated by dioscin; ( B ) ROS generation in SiHa cell treated by dioscin; ( C ) Effects of dioscin on Ca 2+ level in HeLa cells by flow cytometry; ( D ) Effects of dioscin on Ca 2+ level in SiHa cells by flow cytometry. Data are presented as mean ± SD ( n = 5). * p

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques: Flow Cytometry, Cytometry

    Dioscin inhibited cell viability and induced ultrastructure changes. ( A ) Inhibition effects of dioscin on HeLa cells; ( B ) Inhibition effects of dioscin on SiHa cells; ( C ) Effects of dioscin on the ultrastructure of HeLa cells by TEM assay; ( D ) Effects of dioscin on the ultrastructure of SiHa cells by TEM assay. Data are presented as mean ± SD ( n = 5). ** p

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Dioscin inhibited cell viability and induced ultrastructure changes. ( A ) Inhibition effects of dioscin on HeLa cells; ( B ) Inhibition effects of dioscin on SiHa cells; ( C ) Effects of dioscin on the ultrastructure of HeLa cells by TEM assay; ( D ) Effects of dioscin on the ultrastructure of SiHa cells by TEM assay. Data are presented as mean ± SD ( n = 5). ** p

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques: Inhibition, Transmission Electron Microscopy

    Dioscin induced HeLa and SiHa cell apoptosis. ( A ) Dioscin caused apoptosis in HeLa cells by flow cytometric analysis with Annexin V-FITC and PI-staining; ( B ) Dioscin caused apoptosis in SiHa cells by flow cytometric analysis with Annexin V-FITC and PI-staining. Data are presented as mean ± SD ( n = 3). * p

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Dioscin induced HeLa and SiHa cell apoptosis. ( A ) Dioscin caused apoptosis in HeLa cells by flow cytometric analysis with Annexin V-FITC and PI-staining; ( B ) Dioscin caused apoptosis in SiHa cells by flow cytometric analysis with Annexin V-FITC and PI-staining. Data are presented as mean ± SD ( n = 3). * p

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques: Flow Cytometry, Staining

    Dioscin adjusted cell apoptosis signaling pathway. ( A ) Effects of dioscin on the protein levels of Bcl-2, Bcl-xl, Bax, Bak, Bid, p53, caspase-3 and caspase-9 in HeLa cell; ( B ) Effects of dioscin on the protein levels of Bcl-2, Bcl-xl, Bax, Bak, Bid, p53, caspase-3 and caspase-9 in SiHa cell.

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Dioscin adjusted cell apoptosis signaling pathway. ( A ) Effects of dioscin on the protein levels of Bcl-2, Bcl-xl, Bax, Bak, Bid, p53, caspase-3 and caspase-9 in HeLa cell; ( B ) Effects of dioscin on the protein levels of Bcl-2, Bcl-xl, Bax, Bak, Bid, p53, caspase-3 and caspase-9 in SiHa cell.

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques:

    Morphological changes of SiHa cells treated by dioscin.

    Journal: Molecules

    Article Title: Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway

    doi: 10.3390/molecules21060730

    Figure Lengend Snippet: Morphological changes of SiHa cells treated by dioscin.

    Article Snippet: Detection of Cell Apoptosis and Intracellular Ca2+ Release HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry (Becton-Dickinson).

    Techniques:

    A) % Viability and B) % Cell proliferation upon overexpression of Robo 4 in PC3, DU145 and LNCaP cells; data from ≥3 independent experiments, *p

    Journal: International Journal of Medical Sciences

    Article Title: Robo 4 - the double-edged sword in prostate cancer: impact on cancer cell aggressiveness and tumor vasculature

    doi: 10.7150/ijms.28735

    Figure Lengend Snippet: A) % Viability and B) % Cell proliferation upon overexpression of Robo 4 in PC3, DU145 and LNCaP cells; data from ≥3 independent experiments, *p

    Article Snippet: Flow cytometry Cells were seeded in 6-well plates and transfected with overexpression plasmids or siRNA as described above for 96 h. Afterwards cells were trypsinized and cell pellets were re-suspended in propidium iodide (PI) buffer (0.2% Triton-X-100, 2 ng/mL Na-Citrate, and 0.1 mg/mL PI) and kept light-protected at 4°C for 1 h. Apoptosis was analyzed measuring subG1 peak using FACS Calibur (Becton Dickinson).

    Techniques: Over Expression

    BMA097 inhibits proliferation and induces apoptosis in breast cancer cells (A) Survival assay. Human breast cancer cells were treated with BMA097 at different concentrations for various times followed by MTT assay. (B–D) Apoptosis assay. Human breast cancer cells were treated with BMA097 at different concentrations (B and D) or at 2 μM for different times (C) followed by Annexin V/PI (B–C) or JC-1 staining (D) and FACS analysis of apoptosis and mitochondrial membrane potential (Δψ m ). (* p

    Journal: Oncogene

    Article Title: Novel Synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

    doi: 10.1038/s41388-017-0076-0

    Figure Lengend Snippet: BMA097 inhibits proliferation and induces apoptosis in breast cancer cells (A) Survival assay. Human breast cancer cells were treated with BMA097 at different concentrations for various times followed by MTT assay. (B–D) Apoptosis assay. Human breast cancer cells were treated with BMA097 at different concentrations (B and D) or at 2 μM for different times (C) followed by Annexin V/PI (B–C) or JC-1 staining (D) and FACS analysis of apoptosis and mitochondrial membrane potential (Δψ m ). (* p

    Article Snippet: Briefly, 3×105 cells/well were seeded in 6-well plates and treated with BMA097 at different concentrations for various times followed by staining with annexin V-FITC and propidium iodide (PI) and FACS analysis using a flow cytometer (Becton Dickinson, USA).

    Techniques: Clonogenic Cell Survival Assay, MTT Assay, Apoptosis Assay, Staining, FACS

    BMA097 inhibition of STAT3 activity and expression of STAT3 target genes (A and B) Dose response and time course of BMA097 inhibition of STAT3-dependent luciferase expression. MDA-MB-231 cells with stable expression of STAT3-dependent luciferase were incubated with BMA097 at different concentrations (A) or at 2 μM for different times (B) followed by determination of luciferase activity. (C) BMA097 inhibition of STAT3 downstream target gene expression. MDA-MB-231 cells were treated without or with different concentrations of BMA097 followed by real-time RT-PCR analysis of mRNAs encoding STAT3, cyclin D1 and Bcl-xL. GAPDH was used as an internal control. (n=3; * p

    Journal: Oncogene

    Article Title: Novel Synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

    doi: 10.1038/s41388-017-0076-0

    Figure Lengend Snippet: BMA097 inhibition of STAT3 activity and expression of STAT3 target genes (A and B) Dose response and time course of BMA097 inhibition of STAT3-dependent luciferase expression. MDA-MB-231 cells with stable expression of STAT3-dependent luciferase were incubated with BMA097 at different concentrations (A) or at 2 μM for different times (B) followed by determination of luciferase activity. (C) BMA097 inhibition of STAT3 downstream target gene expression. MDA-MB-231 cells were treated without or with different concentrations of BMA097 followed by real-time RT-PCR analysis of mRNAs encoding STAT3, cyclin D1 and Bcl-xL. GAPDH was used as an internal control. (n=3; * p

    Article Snippet: Briefly, 3×105 cells/well were seeded in 6-well plates and treated with BMA097 at different concentrations for various times followed by staining with annexin V-FITC and propidium iodide (PI) and FACS analysis using a flow cytometer (Becton Dickinson, USA).

    Techniques: Inhibition, Activity Assay, Expressing, Luciferase, Multiple Displacement Amplification, Incubation, Quantitative RT-PCR

    Effect of BMA097 on STAT3 phosphorylation and activation (A–B) BMA097 inhibition of constitutive STAT3 phosphorylation. MDA-MB-231 cells were treated with different concentrations of BMA097 for 24 hrs (A and D) or at 2 μM for different times (B) followed by Western blot analysis of total and phosphorylated STAT3. STAT3 downstream target proteins Bcl-xL and Cyclin D1 were also tested. Actin was used as a loading control for Western blot. (C) BMA097 inhibition of IL-6-induced STAT3 phosphorylation. Serum-starved MDA-MB-231 cells were pre-treated with BMA097 and then with IL-6 followed by Western blot analysis of total and Tyr 705 -phosphorylated STAT3. (D–E) BMA097 effect on pSTAT3 subcellular localization. MDA-MB-231 cells were treated without or with BMA097 for 24 hrs followed by immunofluorescence staining or fractionation and Western blot analysis of Tyr 705 -phosphorylated STAT3. DAPI was used to counterstain nuclei in panel D. Histone and actin were used as markers for nuclear and cytoplasmic fractions, respectively, in panel E. (F–G) Effect of BMA097 on STAT3 dimerization. MDA-MB-231 cells were treated with BMA097 as described in panel A followed by separation using non-denaturing PAGE and Western blot analysis of STAT3. (H) Effect of BMA097 on JAK2 activation. MDA-MB-231 cells were treated with BMA097 followed by Western blot analysis of total and Tyr 1007/1008 -phosphorylated JAK2.

    Journal: Oncogene

    Article Title: Novel Synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

    doi: 10.1038/s41388-017-0076-0

    Figure Lengend Snippet: Effect of BMA097 on STAT3 phosphorylation and activation (A–B) BMA097 inhibition of constitutive STAT3 phosphorylation. MDA-MB-231 cells were treated with different concentrations of BMA097 for 24 hrs (A and D) or at 2 μM for different times (B) followed by Western blot analysis of total and phosphorylated STAT3. STAT3 downstream target proteins Bcl-xL and Cyclin D1 were also tested. Actin was used as a loading control for Western blot. (C) BMA097 inhibition of IL-6-induced STAT3 phosphorylation. Serum-starved MDA-MB-231 cells were pre-treated with BMA097 and then with IL-6 followed by Western blot analysis of total and Tyr 705 -phosphorylated STAT3. (D–E) BMA097 effect on pSTAT3 subcellular localization. MDA-MB-231 cells were treated without or with BMA097 for 24 hrs followed by immunofluorescence staining or fractionation and Western blot analysis of Tyr 705 -phosphorylated STAT3. DAPI was used to counterstain nuclei in panel D. Histone and actin were used as markers for nuclear and cytoplasmic fractions, respectively, in panel E. (F–G) Effect of BMA097 on STAT3 dimerization. MDA-MB-231 cells were treated with BMA097 as described in panel A followed by separation using non-denaturing PAGE and Western blot analysis of STAT3. (H) Effect of BMA097 on JAK2 activation. MDA-MB-231 cells were treated with BMA097 followed by Western blot analysis of total and Tyr 1007/1008 -phosphorylated JAK2.

    Article Snippet: Briefly, 3×105 cells/well were seeded in 6-well plates and treated with BMA097 at different concentrations for various times followed by staining with annexin V-FITC and propidium iodide (PI) and FACS analysis using a flow cytometer (Becton Dickinson, USA).

    Techniques: Activation Assay, Inhibition, Multiple Displacement Amplification, Western Blot, Immunofluorescence, Staining, Fractionation, Polyacrylamide Gel Electrophoresis

    Binding of BMA097 to the SH2 domain of STAT3 (A–B) The predicted BMA097 -binding pocket (A) and binding mode (B) in the SH2 domain of STAT3. Molecular surface in (A) is colored by dodger blue for the most hydrophilic to orange red for the most hydrophobic. The green lines in (B) indicate H-bonds. (C) Pull-down assay. Total lysate from MDA-MB-231 cells were subjected to pull-down assay by CNBr-immobilized BMA097 , an irrelevant compound (IC) or vehicle control followed by Western blot analysis of pull-down materials using STAT3 antibody. (D–E) Competition of STAT3-binding to immobilized BMA097 (D) or elution of STAT3 bound to the immobilized BMA097 (E) by excess free BMA097, DMSO vehicle control, or an irrelevant negative control compound (IC). (F) Schematic domain structures of STAT3 and of recombinant proteins. NTD, amino terminal domain; CCD, coiled coil domain; DBD, DNA-binding domain; LD, linker domain; SH2, Src homology 2 domain; TAD, transactivation domain. (G) Pull-down assay of purified recombinant STAT3 proteins with different deletions by immobilized BMA097.

    Journal: Oncogene

    Article Title: Novel Synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

    doi: 10.1038/s41388-017-0076-0

    Figure Lengend Snippet: Binding of BMA097 to the SH2 domain of STAT3 (A–B) The predicted BMA097 -binding pocket (A) and binding mode (B) in the SH2 domain of STAT3. Molecular surface in (A) is colored by dodger blue for the most hydrophilic to orange red for the most hydrophobic. The green lines in (B) indicate H-bonds. (C) Pull-down assay. Total lysate from MDA-MB-231 cells were subjected to pull-down assay by CNBr-immobilized BMA097 , an irrelevant compound (IC) or vehicle control followed by Western blot analysis of pull-down materials using STAT3 antibody. (D–E) Competition of STAT3-binding to immobilized BMA097 (D) or elution of STAT3 bound to the immobilized BMA097 (E) by excess free BMA097, DMSO vehicle control, or an irrelevant negative control compound (IC). (F) Schematic domain structures of STAT3 and of recombinant proteins. NTD, amino terminal domain; CCD, coiled coil domain; DBD, DNA-binding domain; LD, linker domain; SH2, Src homology 2 domain; TAD, transactivation domain. (G) Pull-down assay of purified recombinant STAT3 proteins with different deletions by immobilized BMA097.

    Article Snippet: Briefly, 3×105 cells/well were seeded in 6-well plates and treated with BMA097 at different concentrations for various times followed by staining with annexin V-FITC and propidium iodide (PI) and FACS analysis using a flow cytometer (Becton Dickinson, USA).

    Techniques: Binding Assay, Pull Down Assay, Multiple Displacement Amplification, Western Blot, Negative Control, Recombinant, Purification

    BMA097 inhibits xenograft breast tumor growth and STAT3 phosphorylation and induces cancer cell apoptosis in vivo (A) Volume of xenograft tumors and body weight of mice following treatments. (B–C) Wet weight and gross anatomy of final dissected xenograft tumor masses. (n= 5; *p

    Journal: Oncogene

    Article Title: Novel Synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

    doi: 10.1038/s41388-017-0076-0

    Figure Lengend Snippet: BMA097 inhibits xenograft breast tumor growth and STAT3 phosphorylation and induces cancer cell apoptosis in vivo (A) Volume of xenograft tumors and body weight of mice following treatments. (B–C) Wet weight and gross anatomy of final dissected xenograft tumor masses. (n= 5; *p

    Article Snippet: Briefly, 3×105 cells/well were seeded in 6-well plates and treated with BMA097 at different concentrations for various times followed by staining with annexin V-FITC and propidium iodide (PI) and FACS analysis using a flow cytometer (Becton Dickinson, USA).

    Techniques: In Vivo, Mouse Assay

    Synthesis of BMA analogues and their effects on STAT3-dependent luciferase activity (A) Synthesis of BMA analogues. a. NaH, CH 3 CH 2 I, THF: 1 95% yield; b. NaH, Br(CH 2 ) n CN, MeCN: 2a 94% yield, 3a 57% yield, 4a 83% yield; c. (i) (COCl) 2 , CH 2 Cl 2 , (ii) (CH 3 CH 2 ) 3 N, CH 2 Cl 2 : 2b 23% yield, 3b 45% yield, 4b 51% yield, 5b 44% yield, 6b 32% yield, 7b 39% yield; d. HMDS, MeOH, DMF: BMA016 95% yield, BMA019 98% yield, 4c 95% yield, BMA097 91% yield, BMA100 90% yield, 7c 90% yield; e. 37% HCHO, NaHCO 3 , 85°C: BMA088 96% yield, : BMA127 95% yiled; f. NaH, Br(CH 2 ) n CN, THF: 5a 30% yield, 6a 83% yiled; g. NaH, C 6 H 5 CH 2 Br, DMF: 7a 99% yiled; h. O 2 , DMSO, t -BuOK, THF: 7d 89% yiled. (B) Effect of BMA analogues on STAT3-dependent luciferase activity. Stable MDA-MB-231 cells expressing STAT3-dependent luciferase reporter were treated with synthetic BMA analogues or DMSO control followed by determination of luciferase activity.

    Journal: Oncogene

    Article Title: Novel Synthetic bisindolylmaleimide alkaloids inhibit STAT3 activation by binding to the SH2 domain and suppress breast xenograft tumor growth

    doi: 10.1038/s41388-017-0076-0

    Figure Lengend Snippet: Synthesis of BMA analogues and their effects on STAT3-dependent luciferase activity (A) Synthesis of BMA analogues. a. NaH, CH 3 CH 2 I, THF: 1 95% yield; b. NaH, Br(CH 2 ) n CN, MeCN: 2a 94% yield, 3a 57% yield, 4a 83% yield; c. (i) (COCl) 2 , CH 2 Cl 2 , (ii) (CH 3 CH 2 ) 3 N, CH 2 Cl 2 : 2b 23% yield, 3b 45% yield, 4b 51% yield, 5b 44% yield, 6b 32% yield, 7b 39% yield; d. HMDS, MeOH, DMF: BMA016 95% yield, BMA019 98% yield, 4c 95% yield, BMA097 91% yield, BMA100 90% yield, 7c 90% yield; e. 37% HCHO, NaHCO 3 , 85°C: BMA088 96% yield, : BMA127 95% yiled; f. NaH, Br(CH 2 ) n CN, THF: 5a 30% yield, 6a 83% yiled; g. NaH, C 6 H 5 CH 2 Br, DMF: 7a 99% yiled; h. O 2 , DMSO, t -BuOK, THF: 7d 89% yiled. (B) Effect of BMA analogues on STAT3-dependent luciferase activity. Stable MDA-MB-231 cells expressing STAT3-dependent luciferase reporter were treated with synthetic BMA analogues or DMSO control followed by determination of luciferase activity.

    Article Snippet: Briefly, 3×105 cells/well were seeded in 6-well plates and treated with BMA097 at different concentrations for various times followed by staining with annexin V-FITC and propidium iodide (PI) and FACS analysis using a flow cytometer (Becton Dickinson, USA).

    Techniques: Luciferase, Activity Assay, Multiple Displacement Amplification, Expressing

    SCH-479833 and SCH-527123 inhibit melanoma cell motility and invasion. Cells were plated on noncoated or Matrigel-coated membranes for motility ( A and B ) and invasion ( C and D ) assays and incubated for overnight. Serum-free medium containing 10 ng/mL CXCL-8 and SCH-479833 or SCH-527123 (10 ng/mL) or HPβCD was added to the lower chamber.The cells that did not migrate through the Matrigel and/or pores in the membrane were removed, and cells on the other side of the membrane were stained and photographed at ×200 magnification. Cells were counted in 10 random fields (×200) and expressed as the average number of cells per field of view. Number ± SE of migrated cells. Representative of three experiments done in triplicate. *, P

    Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

    Article Title: Small-Molecule Antagonists for CXCR2 and CXCR1 Inhibit Human Melanoma Growth by Decreasing Tumor Cell Proliferation, Survival, and Angiogenesis

    doi: 10.1158/1078-0432.CCR-08-2387

    Figure Lengend Snippet: SCH-479833 and SCH-527123 inhibit melanoma cell motility and invasion. Cells were plated on noncoated or Matrigel-coated membranes for motility ( A and B ) and invasion ( C and D ) assays and incubated for overnight. Serum-free medium containing 10 ng/mL CXCL-8 and SCH-479833 or SCH-527123 (10 ng/mL) or HPβCD was added to the lower chamber.The cells that did not migrate through the Matrigel and/or pores in the membrane were removed, and cells on the other side of the membrane were stained and photographed at ×200 magnification. Cells were counted in 10 random fields (×200) and expressed as the average number of cells per field of view. Number ± SE of migrated cells. Representative of three experiments done in triplicate. *, P

    Article Snippet: To investigate the effect of SCH-479833 or SCH-527123 on melanoma cell migration, cells (1 × 106 per well) in serum-free medium were plated in the top chamber of noncoated polyethylene terephthalate membranes (6-well insert, 8 Am pore size; Becton Dickinson), whereas for the invasion assay cells (1,000 per well) were plated onto Matrigel-coated Transwell chambers (24-well insert; 8 Am pore size; Corning Costar) with medium (serum-free).

    Techniques: Incubation, Staining