alexa fluor 647 cleaved caspase 3 rabbit mab asp175  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc alexa fluor 647 cleaved caspase 3 rabbit mab asp175
    Alexa Fluor 647 Cleaved Caspase 3 Rabbit Mab Asp175, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/alexa fluor 647 cleaved caspase 3 rabbit mab asp175/product/Cell Signaling Technology Inc
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    alexa fluor 647 cleaved caspase 3 rabbit mab asp175  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc alexa fluor 647 cleaved caspase 3 rabbit mab asp175
    Neuro-2A cultures were infected with dLAT2903, McKrae, and ΔsncRNA1&2 viruses (10 pfu/cell for 24 hr) or mock infected. ( A) Detection of cleaved <t>caspase-3</t> in infected cells . At 24 hr PI, cells were harvested and reacted with anti-cleaved capase-3 antibody and FACS analysis was performed for expression of caspase-3 (see ). Experiments were repeated twice; and ( B) Quantification of FACS analysis from A . Percent of cleaved capase-3 positive cells for each infected or mock infected cells were counted. Each bar represents the mean ± SEM of cleaved capase-3-positive cells (N = 6).
    Alexa Fluor 647 Cleaved Caspase 3 Rabbit Mab Asp175, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The anti-apoptotic function of HSV-1 LAT in neuronal cell cultures but not its function during reactivation correlates with expression of two small non-coding RNAs, sncRNA1&2"

    Article Title: The anti-apoptotic function of HSV-1 LAT in neuronal cell cultures but not its function during reactivation correlates with expression of two small non-coding RNAs, sncRNA1&2

    Journal: PLOS Pathogens

    doi: 10.1371/journal.ppat.1012307

    Neuro-2A cultures were infected with dLAT2903, McKrae, and ΔsncRNA1&2 viruses (10 pfu/cell for 24 hr) or mock infected. ( A) Detection of cleaved caspase-3 in infected cells . At 24 hr PI, cells were harvested and reacted with anti-cleaved capase-3 antibody and FACS analysis was performed for expression of caspase-3 (see ). Experiments were repeated twice; and ( B) Quantification of FACS analysis from A . Percent of cleaved capase-3 positive cells for each infected or mock infected cells were counted. Each bar represents the mean ± SEM of cleaved capase-3-positive cells (N = 6).
    Figure Legend Snippet: Neuro-2A cultures were infected with dLAT2903, McKrae, and ΔsncRNA1&2 viruses (10 pfu/cell for 24 hr) or mock infected. ( A) Detection of cleaved caspase-3 in infected cells . At 24 hr PI, cells were harvested and reacted with anti-cleaved capase-3 antibody and FACS analysis was performed for expression of caspase-3 (see ). Experiments were repeated twice; and ( B) Quantification of FACS analysis from A . Percent of cleaved capase-3 positive cells for each infected or mock infected cells were counted. Each bar represents the mean ± SEM of cleaved capase-3-positive cells (N = 6).

    Techniques Used: Infection, Expressing

    alexa fluor 647 cleaved caspase 3 asp175  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc alexa fluor 647 cleaved caspase 3 asp175
    4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B . Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved <t>caspase</t> <t>3</t> (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D . Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F . ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2 + , red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G . Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)
    Alexa Fluor 647 Cleaved Caspase 3 Asp175, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells"

    Article Title: The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells

    Journal: Virulence

    doi: 10.1080/21505594.2021.1964329

    4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B . Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved caspase 3 (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D . Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F . ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2 + , red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G . Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)
    Figure Legend Snippet: 4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B . Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved caspase 3 (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D . Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F . ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2 + , red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G . Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)

    Techniques Used: Infection, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Luminex, Cell Culture

    anti cleaved caspase 3 alexa fluor 647  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc anti cleaved caspase 3 alexa fluor 647
    a Western blot analysis shows that KRAS G12V overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. b Quantification of KRAS G12V protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown ( n = 2–3 per group). c Representative growth curve of CaCo2 cells after KRAS G12V induction shows no difference in growth with or without KRAS G12V expression ( n = 6 technical replicates; error bars = SEM). d Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. e Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The y -axis depicts the log 2 fold change of shRNA abundance. The x -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS G12V . f Western blots showing MCM7 expression after knockdown. KRAS G12V and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. g , h Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS G12V expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown ( n = 3–4 per group). Student’s t -test (two-sided); NS = not significant; * p < 0.05; ** p < 0.01. i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS G12V and ± MCM7.sh3. KRAS G12V expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS G12V reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM ( n = 3 per group). Student’s t -test (two-sided); NS = not significant; *** p < 0.001. j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS G12V . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. k FACS analysis of cleaved <t>Caspase</t> <t>3</t> (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS G12V and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 + cells ± SEM is shown ( n = 4 per group). Student’s t -test (two-sided); *** p < 0.001; **** p < 0.0001.
    Anti Cleaved Caspase 3 Alexa Fluor 647, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe"

    Article Title: Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

    Journal: Cell Death & Disease

    doi: 10.1038/s41419-020-2704-9

    a Western blot analysis shows that KRAS G12V overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. b Quantification of KRAS G12V protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown ( n = 2–3 per group). c Representative growth curve of CaCo2 cells after KRAS G12V induction shows no difference in growth with or without KRAS G12V expression ( n = 6 technical replicates; error bars = SEM). d Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. e Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The y -axis depicts the log 2 fold change of shRNA abundance. The x -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS G12V . f Western blots showing MCM7 expression after knockdown. KRAS G12V and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. g , h Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS G12V expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown ( n = 3–4 per group). Student’s t -test (two-sided); NS = not significant; * p < 0.05; ** p < 0.01. i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS G12V and ± MCM7.sh3. KRAS G12V expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS G12V reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM ( n = 3 per group). Student’s t -test (two-sided); NS = not significant; *** p < 0.001. j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS G12V . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. k FACS analysis of cleaved Caspase 3 (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS G12V and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 + cells ± SEM is shown ( n = 4 per group). Student’s t -test (two-sided); *** p < 0.001; **** p < 0.0001.
    Figure Legend Snippet: a Western blot analysis shows that KRAS G12V overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. b Quantification of KRAS G12V protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown ( n = 2–3 per group). c Representative growth curve of CaCo2 cells after KRAS G12V induction shows no difference in growth with or without KRAS G12V expression ( n = 6 technical replicates; error bars = SEM). d Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. e Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The y -axis depicts the log 2 fold change of shRNA abundance. The x -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS G12V . f Western blots showing MCM7 expression after knockdown. KRAS G12V and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. g , h Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS G12V expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown ( n = 3–4 per group). Student’s t -test (two-sided); NS = not significant; * p < 0.05; ** p < 0.01. i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS G12V and ± MCM7.sh3. KRAS G12V expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS G12V reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM ( n = 3 per group). Student’s t -test (two-sided); NS = not significant; *** p < 0.001. j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS G12V . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. k FACS analysis of cleaved Caspase 3 (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS G12V and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 + cells ± SEM is shown ( n = 4 per group). Student’s t -test (two-sided); *** p < 0.001; **** p < 0.0001.

    Techniques Used: Western Blot, Over Expression, Activation Assay, Expressing, Mutagenesis, shRNA, Sequencing, Plasmid Preparation, Staining

    caspase 3 alexa fluor 647  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc caspase 3 alexa fluor 647
    Increased apoptosis in retinas from Bcl-2 PC and Bcl-2 AC mice. Retinas from P14 Bcl-2 Flox/Flox , Bcl-2 PC and Bcl-2 AC mice were wholemount stained with anti-cleaved <t>caspase</t> <t>3</t> (white), anti-ERG (green), anti-Pax2 (red) and Isolectin-B4-FITC (blue). The cleaved caspase-3 positive cells associated with the vasculature were quantified throughout the entire retina that were anti-ERG positive (endothelial cells), anti-Pax-2 positive (astrocytes) or non-labeled (pericytes). Arrows point to apoptotic endothelial cell (green), astrocyte (red) and pericyte (white). The inset is a 200% magnification of the boxed areas of interest. Scale bar = 100 µm. The mean number of cleaved caspase 3 positive endothelial cells, pericytes and astrocytes associated with the vasculature of each retina is denoted in the bar graph. Please note macrophages distinguished by their intense staining and large size, were not counted in the total number of capase 3 positive cells (n = 6, ***P < 0.001, ****P < 0.0001).
    Caspase 3 Alexa Fluor 647, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Bcl-2 Expression in Pericytes and Astrocytes Impacts Vascular Development and Homeostasis"

    Article Title: Bcl-2 Expression in Pericytes and Astrocytes Impacts Vascular Development and Homeostasis

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-45915-4

    Increased apoptosis in retinas from Bcl-2 PC and Bcl-2 AC mice. Retinas from P14 Bcl-2 Flox/Flox , Bcl-2 PC and Bcl-2 AC mice were wholemount stained with anti-cleaved caspase 3 (white), anti-ERG (green), anti-Pax2 (red) and Isolectin-B4-FITC (blue). The cleaved caspase-3 positive cells associated with the vasculature were quantified throughout the entire retina that were anti-ERG positive (endothelial cells), anti-Pax-2 positive (astrocytes) or non-labeled (pericytes). Arrows point to apoptotic endothelial cell (green), astrocyte (red) and pericyte (white). The inset is a 200% magnification of the boxed areas of interest. Scale bar = 100 µm. The mean number of cleaved caspase 3 positive endothelial cells, pericytes and astrocytes associated with the vasculature of each retina is denoted in the bar graph. Please note macrophages distinguished by their intense staining and large size, were not counted in the total number of capase 3 positive cells (n = 6, ***P < 0.001, ****P < 0.0001).
    Figure Legend Snippet: Increased apoptosis in retinas from Bcl-2 PC and Bcl-2 AC mice. Retinas from P14 Bcl-2 Flox/Flox , Bcl-2 PC and Bcl-2 AC mice were wholemount stained with anti-cleaved caspase 3 (white), anti-ERG (green), anti-Pax2 (red) and Isolectin-B4-FITC (blue). The cleaved caspase-3 positive cells associated with the vasculature were quantified throughout the entire retina that were anti-ERG positive (endothelial cells), anti-Pax-2 positive (astrocytes) or non-labeled (pericytes). Arrows point to apoptotic endothelial cell (green), astrocyte (red) and pericyte (white). The inset is a 200% magnification of the boxed areas of interest. Scale bar = 100 µm. The mean number of cleaved caspase 3 positive endothelial cells, pericytes and astrocytes associated with the vasculature of each retina is denoted in the bar graph. Please note macrophages distinguished by their intense staining and large size, were not counted in the total number of capase 3 positive cells (n = 6, ***P < 0.001, ****P < 0.0001).

    Techniques Used: Staining, Labeling

    rabbit cleaved caspase 3 asp175 d3e9 alexa fluor 647 conjugate  (Cell Signaling Technology Inc)


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    Cell Signaling Technology Inc rabbit cleaved caspase 3 asp175 d3e9 alexa fluor 647 conjugate
    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved <t>caspase-3</t> staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3
    Rabbit Cleaved Caspase 3 Asp175 D3e9 Alexa Fluor 647 Conjugate, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity"

    Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.11.035

    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved caspase-3 staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3
    Figure Legend Snippet: (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved caspase-3 staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3

    Techniques Used: Labeling, Staining

    KEY RESOURCES TABLE
    Figure Legend Snippet: KEY RESOURCES TABLE

    Techniques Used: Recombinant, Single Cell Gel Electrophoresis, Protein Quantitation, Software

    cleaved caspase 3 rabbit mab alexa fluor 647 conjugate  (Cell Signaling Technology Inc)


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    4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B . Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved <t>caspase</t> <t>3</t> (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D . Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F . ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2 + , red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G . Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)
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    a Western blot analysis shows that KRAS G12V overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. b Quantification of KRAS G12V protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown ( n = 2–3 per group). c Representative growth curve of CaCo2 cells after KRAS G12V induction shows no difference in growth with or without KRAS G12V expression ( n = 6 technical replicates; error bars = SEM). d Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. e Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The y -axis depicts the log 2 fold change of shRNA abundance. The x -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS G12V . f Western blots showing MCM7 expression after knockdown. KRAS G12V and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. g , h Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS G12V expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown ( n = 3–4 per group). Student’s t -test (two-sided); NS = not significant; * p < 0.05; ** p < 0.01. i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS G12V and ± MCM7.sh3. KRAS G12V expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS G12V reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM ( n = 3 per group). Student’s t -test (two-sided); NS = not significant; *** p < 0.001. j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS G12V . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. k FACS analysis of cleaved <t>Caspase</t> <t>3</t> (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS G12V and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 + cells ± SEM is shown ( n = 4 per group). Student’s t -test (two-sided); *** p < 0.001; **** p < 0.0001.
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    Increased apoptosis in retinas from Bcl-2 PC and Bcl-2 AC mice. Retinas from P14 Bcl-2 Flox/Flox , Bcl-2 PC and Bcl-2 AC mice were wholemount stained with anti-cleaved <t>caspase</t> <t>3</t> (white), anti-ERG (green), anti-Pax2 (red) and Isolectin-B4-FITC (blue). The cleaved caspase-3 positive cells associated with the vasculature were quantified throughout the entire retina that were anti-ERG positive (endothelial cells), anti-Pax-2 positive (astrocytes) or non-labeled (pericytes). Arrows point to apoptotic endothelial cell (green), astrocyte (red) and pericyte (white). The inset is a 200% magnification of the boxed areas of interest. Scale bar = 100 µm. The mean number of cleaved caspase 3 positive endothelial cells, pericytes and astrocytes associated with the vasculature of each retina is denoted in the bar graph. Please note macrophages distinguished by their intense staining and large size, were not counted in the total number of capase 3 positive cells (n = 6, ***P < 0.001, ****P < 0.0001).
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    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved <t>caspase-3</t> staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3
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    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved <t>caspase-3</t> staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3
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    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved <t>caspase-3</t> staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3
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    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved <t>caspase-3</t> staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3
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    4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B . Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved caspase 3 (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D . Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F . ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2 + , red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G . Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)

    Journal: Virulence

    Article Title: The ApoA-I mimetic peptide 4F attenuates in vitro replication of SARS-CoV-2, associated apoptosis, oxidative stress and inflammation in epithelial cells

    doi: 10.1080/21505594.2021.1964329

    Figure Lengend Snippet: 4 F restricts apoptosis and inflammatory responses associated with SARS-CoV-2 infection in lung epithelial cells. A-D. Vero E6 and Calu3 cells were uninfected (mock) or infected with SARS-CoV-2 (MOI 0.1) and were treated with media alone (vehicle), or 4 F (10 μM) as in methods. Confluent cells were fixed 48 h post-infection (hpi) followed by processing for flow cytometry. A, B . Flow cytometry was used in Vero E6 cells to determine the percent of viable cells positive for cleaved caspase 3 (a) and the median fluorescence intensity (MFI) of cleaved caspase 3 (b) compared to a negative cell population (shown in light gray). Representative data from three independent experiments are shown. C, D . Flow cytometry was used in Calu3 cells to determine the percent of viable cells positive for cleaved caspase 3 (c) and the MFI of cleaved caspase 3 (d) compared to a negative cell population. Representative data from three independent experiments are shown. E, F . ELISA was used to determine protein levels of IL-6 (ng/ml) secreted by Calu3 (e) and Vero-E6 (f) cells 48 hours post SARS-CoV-2 infection. The compared groups were uninfected cells (mock, light gray), cells infected with SARS-CoV-2 (SARS-CoV-2 + , red), and cells infected with SARS-CoV-2 treated with 4 F (light blue). Data represent the mean ± standard error of means (SEM), representing three independent experiments conducted at least in triplicate. The Mann-Whitney was used to compare each group relative to the vehicle control and the p value for this comparison is shown above each column (***p < 0.001). G . Luminex immunoassays were used to determine protein levels of IL-1β, IL-8, IL-10, TNF-α (pg/ml) secreted by Calu-3 cells 48 hours post SARS-CoV-2 infection. The mean value of each measurement (protein levels in cell culture supernatants in pg/ml) was normalized by the mean value of each measurement in the vehicle group and expressed as fold to the mean of the vehicle control group. Data represent the mean ± SEM, representing three independent experiments conducted at least in triplicate. The Kruskal-Wallis statistical test was used to compare 3 groups and the Mann-Whitney was used to compare each group relative to the vehicle control. The p value for this comparison is shown above each column (*p < 0.05, **p < 0.01, ***p < 0.001)

    Article Snippet: The following antibodies were added to each tube and incubated in the dark for 20 minutes on ice: PE/Cyanine7 mouse anti-human CD147 antibody (clone HIM6 from Biolegend, San Diego, CA), mouse anti-human HO-1 antibody (clone HO-1-2 from Enzo Life Sciences) conjugated with Mix-n-Stain CF647 Antibody Labeling Kit from Biotium Inc (Hayward, CA), rabbit anti-human MX1 polyclonal antibody (from Proteintech, 13,750-1-AP), rabbit anti-SARS-CoV-2 (2019-nCoV) Spike S1 Antibody (Clone R007, Cat# 40,150-R007 from Sino Biologicals conjugated with Mix-n-Stain CF488 Antibody Labeling Kit from Biotium Inc (Hayward, CA), mouse anti-SARS-CoV/SARS-CoV-2 spike S antibody (clone 1A9 from GeneTex), Alexa Fluor® 647 Cleaved Caspase-3 (Asp175) (Clone D3E9 from Cell Signaling Technology, Cat# 9602S), Alexa Fluor® 647 Endorepellin/Perlecan/Heparan Sulfate Proteoglycan Antibody (clone A7L6 cat# NBP2-44,448).

    Techniques: Infection, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Luminex, Cell Culture

    a Western blot analysis shows that KRAS G12V overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. b Quantification of KRAS G12V protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown ( n = 2–3 per group). c Representative growth curve of CaCo2 cells after KRAS G12V induction shows no difference in growth with or without KRAS G12V expression ( n = 6 technical replicates; error bars = SEM). d Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. e Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The y -axis depicts the log 2 fold change of shRNA abundance. The x -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS G12V . f Western blots showing MCM7 expression after knockdown. KRAS G12V and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. g , h Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS G12V expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown ( n = 3–4 per group). Student’s t -test (two-sided); NS = not significant; * p < 0.05; ** p < 0.01. i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS G12V and ± MCM7.sh3. KRAS G12V expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS G12V reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM ( n = 3 per group). Student’s t -test (two-sided); NS = not significant; *** p < 0.001. j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS G12V . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. k FACS analysis of cleaved Caspase 3 (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS G12V and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 + cells ± SEM is shown ( n = 4 per group). Student’s t -test (two-sided); *** p < 0.001; **** p < 0.0001.

    Journal: Cell Death & Disease

    Article Title: Reduced replication origin licensing selectively kills KRAS-mutant colorectal cancer cells via mitotic catastrophe

    doi: 10.1038/s41419-020-2704-9

    Figure Lengend Snippet: a Western blot analysis shows that KRAS G12V overexpression is at physiological levels and potent to induce downstream activation of ERK. Vinculin serves as a loading control. b Quantification of KRAS G12V protein expression after 1, 2, and 3 days of doxycycline induction. Mean expression ± SEM is shown ( n = 2–3 per group). c Representative growth curve of CaCo2 cells after KRAS G12V induction shows no difference in growth with or without KRAS G12V expression ( n = 6 technical replicates; error bars = SEM). d Graphical outline of the RNAi screen to identify genetic interactors of mutant KRAS. e Change of shRNA abundance in the shRNA screen after 21 days of culture with and without doxycycline as determined by massive parallel sequencing. Each dot represents a shRNA. The y -axis depicts the log 2 fold change of shRNA abundance. The x -axis shows each shRNA ranked by their fold change. The shRNA MCM7.1879 is specifically depleted when co-expressed with doxycycline induced KRAS G12V . f Western blots showing MCM7 expression after knockdown. KRAS G12V and shRNA expression were induced with doxycycline for 0, 1, 3, 5, or 7 days. MCM7 knockdown was efficient after 3 days of shRNA expression. Vinculin serves as loading control. g , h Clonogenic assays of three non-overlapping shRNAs targeting MCM7 show increased sensitivity of KRAS G12V expressing CaCo2 cells to MCM7 knockdown. Mean percentage of the relative area covered by cells ± SEM is shown ( n = 3–4 per group). Student’s t -test (two-sided); NS = not significant; * p < 0.05; ** p < 0.01. i Soft agar assays showing anchorage-independent growth in CaCo2 cells ± induced KRAS G12V and ± MCM7.sh3. KRAS G12V expression increases colony formation in CaCo2 cells about five fold. MCM7 knockdown has no detectable effect on cells expressing an empty control vector, whereas cells expressing additionally KRAS G12V reduce colony formation significantly. Mean fold change of colonies formed after five weeks of culture is shown ± SEM ( n = 3 per group). Student’s t -test (two-sided); NS = not significant; *** p < 0.001. j Western blots show an increase in cleaved PARP (clPARP) staining after five and seven days of MCM7 knockdown specifically in CaCo2 cells expressing KRAS G12V . ß-Tubulin and total PARP serve as a loading control, the cleaved PARP is indicated by an arrow. k FACS analysis of cleaved Caspase 3 (clCaspase 3) reveals a significant increase of apoptosis in cells co-expressing KRAS G12V and MCM7.sh3 compared to cells expressing MCM7.sh3 alone. Mean percentage of clCaspase 3 + cells ± SEM is shown ( n = 4 per group). Student’s t -test (two-sided); *** p < 0.001; **** p < 0.0001.

    Article Snippet: Subsequently, the fixed cells were washed twice with incubation buffer (0.5% BSA in PBS) and stained for 1 h with anti-Cleaved Caspase-3 Alexa Fluor® 647 (CST #9602) antibody at ambient temperature.

    Techniques: Western Blot, Over Expression, Activation Assay, Expressing, Mutagenesis, shRNA, Sequencing, Plasmid Preparation, Staining

    Increased apoptosis in retinas from Bcl-2 PC and Bcl-2 AC mice. Retinas from P14 Bcl-2 Flox/Flox , Bcl-2 PC and Bcl-2 AC mice were wholemount stained with anti-cleaved caspase 3 (white), anti-ERG (green), anti-Pax2 (red) and Isolectin-B4-FITC (blue). The cleaved caspase-3 positive cells associated with the vasculature were quantified throughout the entire retina that were anti-ERG positive (endothelial cells), anti-Pax-2 positive (astrocytes) or non-labeled (pericytes). Arrows point to apoptotic endothelial cell (green), astrocyte (red) and pericyte (white). The inset is a 200% magnification of the boxed areas of interest. Scale bar = 100 µm. The mean number of cleaved caspase 3 positive endothelial cells, pericytes and astrocytes associated with the vasculature of each retina is denoted in the bar graph. Please note macrophages distinguished by their intense staining and large size, were not counted in the total number of capase 3 positive cells (n = 6, ***P < 0.001, ****P < 0.0001).

    Journal: Scientific Reports

    Article Title: Bcl-2 Expression in Pericytes and Astrocytes Impacts Vascular Development and Homeostasis

    doi: 10.1038/s41598-019-45915-4

    Figure Lengend Snippet: Increased apoptosis in retinas from Bcl-2 PC and Bcl-2 AC mice. Retinas from P14 Bcl-2 Flox/Flox , Bcl-2 PC and Bcl-2 AC mice were wholemount stained with anti-cleaved caspase 3 (white), anti-ERG (green), anti-Pax2 (red) and Isolectin-B4-FITC (blue). The cleaved caspase-3 positive cells associated with the vasculature were quantified throughout the entire retina that were anti-ERG positive (endothelial cells), anti-Pax-2 positive (astrocytes) or non-labeled (pericytes). Arrows point to apoptotic endothelial cell (green), astrocyte (red) and pericyte (white). The inset is a 200% magnification of the boxed areas of interest. Scale bar = 100 µm. The mean number of cleaved caspase 3 positive endothelial cells, pericytes and astrocytes associated with the vasculature of each retina is denoted in the bar graph. Please note macrophages distinguished by their intense staining and large size, were not counted in the total number of capase 3 positive cells (n = 6, ***P < 0.001, ****P < 0.0001).

    Article Snippet: Anti-cleaved caspase-3 Alexa Fluor 647(1:50; Cell Signaling clone D3E9 # 9602) or anti-Ki-67 Alex Fluor 647 (1:50; Cell Signaling clone D3B5 #12075) were diluted in blocking solution with normal rabbit IgG (1:10) and rocked for 48 hours.

    Techniques: Staining, Labeling

    (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved caspase-3 staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3

    Journal: Molecular cell

    Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

    doi: 10.1016/j.molcel.2017.11.035

    Figure Lengend Snippet: (A) Analysis of cell cycle progression in control and DDI1/2 depleted cells following synchronization by 20h 2mM HU treatment and release. 30 min prior to indicated timepoints, cells were labeled with BrdU. Images are representative of at least 3 independent experiments. (B) Analysis of the percentage of apoptotic cells as determined by cleaved caspase-3 staining measured by FACS. U2OS control and DDI1/2 depleted cells with or without knockdown of RTF2 were synchronization by 20 h 2 mM HU treatment and released for indicated times. Error bars represent SEM n=3. (C) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] and newly-fired origins [newly fired origins/(continuing forks plus newly fired origins)] following 20 h of 2 mM HU in control and DDI1/2 knockdown cells. (D) Quantification of the percentage of restarted forks [restarted forks/(restarted forks plus non-restarted forks)] following 4h of 4 mM HU and of fork restart productivity defined by the ratio of CldU length to IdU length of restarted forks following 4h of 4 mM HU in control and DDI1/2 knockdown cells. (E) Analysis of fork processivity defined by the ratio of CldU to IdU label length of continuing forks treated with 0.4 uM aphidicolin during CldU labeling in control and DDI1/2 depleted cells with or without knockdown of RTF2. Each panel includes a schematic for experimental setup. Error bars represent SEM n=3. *p<0.05 **p<0.01 ***p<0.001 ****p<0.0001 by ANOVA. See also Figure S3

    Article Snippet: Rabbit cleaved caspase-3 (Asp175) (D3E9)-Alexa Fluor® 647 Conjugate , Cell Signaling , Cat# 9602.

    Techniques: Labeling, Staining

    KEY RESOURCES TABLE

    Journal: Molecular cell

    Article Title: Removal of RTF2 from stalled replisomes promotes maintenance of genome integrity

    doi: 10.1016/j.molcel.2017.11.035

    Figure Lengend Snippet: KEY RESOURCES TABLE

    Article Snippet: Rabbit cleaved caspase-3 (Asp175) (D3E9)-Alexa Fluor® 647 Conjugate , Cell Signaling , Cat# 9602.

    Techniques: Recombinant, Single Cell Gel Electrophoresis, Protein Quantitation, Software