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  • 99
    Thermo Fisher calcein
    Invasive potential of neuroblastoma cell lines. a. 20 neuroblastoma cell lines were serum-starved for 24 hours, and plated at a seeding density of 0.5 X 10 5 cells per well on to 0.8 micron cell culture inserts, coated with Basement Membrane Extract (BME) coat at 0.5x to 1x dilution. The number of cells crossing the mesh towards RPMI (control) or mRPMI were counted fluorimetrically using <t>calcein</t> AM after 12 hours. Plotted is the average number of cells, for each cell line from 7 experiments, migrating across the mesh. The red line indicates the mean value. Cell lines higher than the mean value were classified as highly invasive and those below the mean value as less invasive cell lines. b. Invasion index was calculated as the ratio of number of cells invading towards mRPMI versus the number of cells invading towards RPMI. Plotted is the mean invasion index from 7 experiments in increasing order. Cell lines with an invasion index of 1 or higher was classified as highly invasive cell lines and those with an invasion index of less than 1 was classified as less invasive.
    Calcein, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore calcein am
    <t>Calcein-AM</t> flux in multidrug-resistant CEM cells. Calcein flux was measured as described in Materials and Methods. Relative fluorescence units (F) (mean ± standard deviation) are shown. Panel A shows the rate of accumulation of calcein in MDR1 + CEM/VBL100 (broken line) and wild-type CEM/WTB (solid line) cells. The effects of reserpine on the calcein flux in CEM/VBL100 and CEM/WTB cells are shown in panels B and C, respectively. Calcein fluorescence of CEM/VBL100 cells was significantly enhanced when incubated with the MDR1 antagonist reserpine (panel B, solid line) compared to CEM/VBL100 cells incubated with saline (panel B, broken line). By contrast, the calcein fluorescence of wild-type CEM/WTB cells was not different when incubated in the presence of either reserpine (hatched line) or saline (solid line). The P values indicate the statistical differences between the slopes of calcein fluorescence in CEM/WTB and in CEM/VBL100 cells (A) or between cultures incubated with reserpine and controls (B and C).
    Calcein Am, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1969 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher calcein am
    DNA damage and cell death in CuONPs-treated HUVECs. ( A ) MTS assay of HUVECs treated with different concentrations of CuONPs (0, 10, 20, or 40 μg/mL) for 24 h. ( B ) Representative FACS data for CuONPs-treated HUVECs after <t>Calcein</t> AM (live cell fluorescent probe) staining. ( C ) Comet assay to evaluate CuONPs-induced DNA damage in HUVECs. ( D ) Immunofluorescence assay of γH2AX foci formation in CuONPs-treated HUVECs. ( E ) Western blotting assay of phospho-ATR, phospho-ATM, phospho-p53 and phospho-H2AX (γH2AX) in HUVECs treated with CuONPs (20 μg/mL) for 0, 1, 3, 6 and 12 h. Actin was used as a loading control. In ( A ), one-way ANOVA followed by Tukey’s test was performed for statistical analysis. In ( B ), unpaired Student’s t -tests were performed for statistical analysis. ** p
    Calcein Am, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 10200 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Dojindo Labs calcein am
    γδ T cells specifically kill Zol-treated HCC cell lines without killing Zol-untreated cells. Bystander cytotoxic activity of γδ T cells was determined in Zol-untreated target cells (HepG2, T2, or PHA blasts). γδ T effector (E) cells (3.0×10 5 cells/well) were co-cultured for 4 h with 5 μM Zol-pretreated (16 h) HepG2 cells (1.0×10 4 cells/well), along with <t>Calcein-AM-labeled,</t> Zol-untreated target (T) cells (1.0×10 4 cells). Zol-pretreated (5 μM, 16 h) HepG2, T2, or PHA blasts served as references. Data represent the mean ± SD of triplicate cultures. Representative results of three independent experiments are shown.
    Calcein Am, supplied by Dojindo Labs, used in various techniques. Bioz Stars score: 94/100, based on 461 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson calcein am
    In vitro crawling on ICAM-1 or ICAM-2. (A). Neutrophils were fluorescently labelled with <t>Calcein-AM</t> and seeded into un-coated (BSA blocked), or ICAM-2- or ICAM-1-coated chamber slides. Following adhesion, anti-MAC-1 mAb (5 µg/ml) was added to some groups. Images were captured at 2-minute intervals and crawling dynamics were analysed using Imaris. Examples show the position of neutrophils at t = 0 in green, and the crawling tracks over the following 40-minute period in black. Neutrophil crawling speed (B) and total crawling distance (C) was quantified on uncoated, ICAM-2- and ICAM-1-coated glass, with or without anti-MAC-1 mAb. Data were obtained from analysis of > 300 crawling cells per group from four or five mice. The black lines show the mean±s.e.m. for all events analysed. *** P
    Calcein Am, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 683 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biotium calcein am
    Misoprostol promotes nuclear calcium accumulation and regulates gene expression. (A) H9c2 cells treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. NLS-R-GECO (red) was used to indicate nuclear calcium content in all conditions. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. (B) Quantification of cells in (A), where red fluorescent signal was normalized to cell area and quantified in 10 random fields. (C) H9c2 cells treated as in (A) and Myc-NFATc3 was transfected into each condition. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc primary antibody (green). Cells were then imaged by standard fluorescence microscopy. (D) Quantification of cells in (C), where the number of cells with nuclear Myc-NFATc3, is presented as a percentage of the number of cells/field in 5 random fields. (E) H9c2 cells treated as in (A), Protein extracts were subjected to nuclear/cytosolic fractionation and were immunoblotted, as indicated. (F) H9c2’s treated as in (A). 2 μM CsA was added to half of the conditions for 48 hours in order to inhibit calcineurin-NFAT signalling. Peredox-mCherry (where Red=NAD + , Green=NADH) was used to indicate cytosolic reduced or oxidized NAD content in all conditions. Cells were imaged by standard fluorescence microscopy. (G) Quantification of cells in (F), where the ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) in 15 random fields across 3 independent experiments. (H) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or myc-NFATc3. Peredox-mCherry was used to indicate cytosolic NAD content. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in > 15 random fields across 6 independent experiments. (I) H9c2’s treated as in (H), cells were stained with Tag-it Violet (blue) and <t>calcein-AM</t> (green) and imaged by standard fluorescence microscopy. (J) Quantification of Tag-it Violet positive cells in (I), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (K) Immunoblot for MEF2C and MEF2A in protein extracts from PVNCs treated as in (A). (L) PVNCs treated as in (A). RNA was isolated and qRT-PCR was performed for BMP-10 expression. All data are represented as mean ± S.E.M. *P
    Calcein Am, supplied by Biotium, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    AnaSpec calcein am
    TGF-β1 pretreated MSCs display higher adhesivity and traction force after removal of stimulus. (A) TGF-β1 pretreated cells display higher adhesivity on both glass and gel. (B) Pretreated MSCs (CM vs. TGF-β1) plated on glass and PA gels were washed after 3 h to remove detached cells. (Red-Actin, Blue- DAPI, Green- <t>Calcein</t> AM). (C) Traction force heat map of CM and TGF-β1 treated MSCs, (D) TGF-β1 pretreated MSCs exerted higher traction force compared to control after 24 h' removal of stimulus. Values given as mean ± s.e.m.; significance is indicated as * ( p
    Calcein Am, supplied by AnaSpec, used in various techniques. Bioz Stars score: 92/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology calcein am
    The ndk null-mutant enhances P. aeruginosa virulence in A549 cells through T3SS activation. Prior to cell infection, the indicated bacterial strains were cultured in LB broth for 12 h. The bacterial pellets and culture supernatants were collected after centrifugation. A549 cells were infected with the indicated strains (MOI = 50). In addition, A549 cells were treated with bacterial culture media 5-fold diluted in DMEM. At 2 h post-infection, the cell viability was detected. ( a ) CCK-8 assay. The percentage of cytotoxicity was calculated according to the following formula: cytotoxicity % = 1 − (OD 450 infected cells /OD 450 sham-infected control ) × 100%. ( b ) <t>Calcein-AM</t> staining. Scale bar: 50 μm. Representative images from triplicate experiments are shown. ( c ) Fluorescence intensity per cell from ( b ) was analyzed by Image pro-Plus software (IPP, edition 6.0) (n cell = 90). Data are expressed as the mean ± SD from three independent experiments. *Indicates P
    Calcein Am, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 148 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Corning Life Sciences calcein am
    Expansion of viable cancer cells is increased in the three-dimensional (3D) cell-sheet model. <t>Calcein-AM</t> (green) and propidium iodide (PI, red) allow observation of viable and dead cells in the cancer spheroid (A) and 3D cell-sheet model (B) with or without CAFs. The spheroid and 3D cell sheet were exposed to vehicle control (ctr), 10 μM sorafenib, or 20 μM cisplatin for 72 h. The area of viable cells was larger in the 3D cell sheet than in the cancer spheroid because of a more peripheral spread of cancer cells in the 3D cell-sheet model. Bars indicate 100 μm.
    Calcein Am, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 91/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore calcein am solution
    Stable knockdown of ALKBH3 and RAD51C results in severe MMS-induced cytotoxicity in PC3 and NCI-H23 cells. ( A ) HEK293T was transiently transfected with shRNA for 72 h. Knockdown of ALKBH3 and RAD51C was confirmed by western blotting with indicated antibodies. Effect of stable knockdown of ALKBH3 and RAD51C on ( B ) PC-3 and ( C ) NCI-H23. Cells with indicated shRNAs were exposed to various concentrations of MMS (0, 50, 100, 200, 300 and 400 μM) for 48 h; then cell viability was evaluated by MTT assay. Error bar represent the means ± SD of percentage cell viability from four representative experiments. ( D ) Analysis of MMS sensitivity using <t>Calcein-AM</t> assay. Representative images of intracellular Calcein AM esterase activity of PC-3 and ( F ) NCI-H23 cells. Cells were treated with MMS(400uM) for 48h and fluorescent images were captured using an inverted microscope. Bar chart represents the percentage (%) of MMS-induced cytotoxicity of ( E ) PC-3 and ( G ) NCI-H23 cells for the indicated shRNAs.
    Calcein Am Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Invasive potential of neuroblastoma cell lines. a. 20 neuroblastoma cell lines were serum-starved for 24 hours, and plated at a seeding density of 0.5 X 10 5 cells per well on to 0.8 micron cell culture inserts, coated with Basement Membrane Extract (BME) coat at 0.5x to 1x dilution. The number of cells crossing the mesh towards RPMI (control) or mRPMI were counted fluorimetrically using calcein AM after 12 hours. Plotted is the average number of cells, for each cell line from 7 experiments, migrating across the mesh. The red line indicates the mean value. Cell lines higher than the mean value were classified as highly invasive and those below the mean value as less invasive cell lines. b. Invasion index was calculated as the ratio of number of cells invading towards mRPMI versus the number of cells invading towards RPMI. Plotted is the mean invasion index from 7 experiments in increasing order. Cell lines with an invasion index of 1 or higher was classified as highly invasive cell lines and those with an invasion index of less than 1 was classified as less invasive.

    Journal: PLoS ONE

    Article Title: Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines

    doi: 10.1371/journal.pone.0120069

    Figure Lengend Snippet: Invasive potential of neuroblastoma cell lines. a. 20 neuroblastoma cell lines were serum-starved for 24 hours, and plated at a seeding density of 0.5 X 10 5 cells per well on to 0.8 micron cell culture inserts, coated with Basement Membrane Extract (BME) coat at 0.5x to 1x dilution. The number of cells crossing the mesh towards RPMI (control) or mRPMI were counted fluorimetrically using calcein AM after 12 hours. Plotted is the average number of cells, for each cell line from 7 experiments, migrating across the mesh. The red line indicates the mean value. Cell lines higher than the mean value were classified as highly invasive and those below the mean value as less invasive cell lines. b. Invasion index was calculated as the ratio of number of cells invading towards mRPMI versus the number of cells invading towards RPMI. Plotted is the mean invasion index from 7 experiments in increasing order. Cell lines with an invasion index of 1 or higher was classified as highly invasive cell lines and those with an invasion index of less than 1 was classified as less invasive.

    Article Snippet: The cells invading across the mesh were harvested using 100 μl of 1x Cell Dissociation Solution (CDS) (Trevigen, Cat no #3455–096–05) containing 1.2 μl of Calcein AM (Molecular Probes, Cat no #C3100MP) solution (1.67 μg/μl in DMSO).

    Techniques: Cell Culture

    In vitro characterization of convertible CAR activity. a Ramos (CD20 + ) target cells were exposed to convertible CAR-CD8 + cells at an E:T of 5:1 and co-cultured with increasing concentrations of rituximab antibody (ADCC-deficient), Rituximab.LC-U2S3 MicAbody, or Trastuzumab.LC-U2S3 MicAbody. After 24 h, supernatants were harvested and IL-2 (solid bars) or IFNγ (hatched bars) quantified by ELISA. b convertible CAR-CD8 + cells were incubated with increasing concentrations of Alexa Fluor 647 conjugated Rituximab.LC-U2S3 for 30 min, the excess washed away, and the MFI quantified by flow cytometry. Red line at 5 nM indicates inflection point at which receptors are maximally occupied. c convertible CAR-CD8 + cells were armed with increasing concentrations of Rituximab.LC-U2S3 as described in ( b ) then co-incubated with calcein-loaded Ramos cells at an E:T of 20:1 for 2 h after which the amount of released calcein was quantified. d iNKG2D.YA-CAR CD8 + cells were pre-armed with 5 nM Rituximab.LC-U2S3, 5 nM Trastuzumab.LC-U2S3, or an equimolar mixture of 2.5 nM of each as described in ( b ) then exposed to calcein-loaded Ramos or CT26-Her2 cells at two indicated E:T ratios. The amount of calcein released was quantified after two hours. Except for b , data are representative of at least two independent experiments and plotted as an average of technical triplicates.

    Journal: Communications Biology

    Article Title: convertibleCARs: A chimeric antigen receptor system for flexible control of activity and antigen targeting

    doi: 10.1038/s42003-020-1021-2

    Figure Lengend Snippet: In vitro characterization of convertible CAR activity. a Ramos (CD20 + ) target cells were exposed to convertible CAR-CD8 + cells at an E:T of 5:1 and co-cultured with increasing concentrations of rituximab antibody (ADCC-deficient), Rituximab.LC-U2S3 MicAbody, or Trastuzumab.LC-U2S3 MicAbody. After 24 h, supernatants were harvested and IL-2 (solid bars) or IFNγ (hatched bars) quantified by ELISA. b convertible CAR-CD8 + cells were incubated with increasing concentrations of Alexa Fluor 647 conjugated Rituximab.LC-U2S3 for 30 min, the excess washed away, and the MFI quantified by flow cytometry. Red line at 5 nM indicates inflection point at which receptors are maximally occupied. c convertible CAR-CD8 + cells were armed with increasing concentrations of Rituximab.LC-U2S3 as described in ( b ) then co-incubated with calcein-loaded Ramos cells at an E:T of 20:1 for 2 h after which the amount of released calcein was quantified. d iNKG2D.YA-CAR CD8 + cells were pre-armed with 5 nM Rituximab.LC-U2S3, 5 nM Trastuzumab.LC-U2S3, or an equimolar mixture of 2.5 nM of each as described in ( b ) then exposed to calcein-loaded Ramos or CT26-Her2 cells at two indicated E:T ratios. The amount of calcein released was quantified after two hours. Except for b , data are representative of at least two independent experiments and plotted as an average of technical triplicates.

    Article Snippet: For calcein-release assays, tumor cells were centrifuged and resuspend in 4 mM probenecid (MP Biomedicals #156370) + 25 µM calcein-AM (Thermo Fisher #C1430) in T cell medium at 1–2 × 106 cells/ml for one hour at 37 °C, washed once, and adjusted to 8 × 105 cells/ml.

    Techniques: In Vitro, Activity Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Incubation, Flow Cytometry

    Calcein-AM flux in multidrug-resistant CEM cells. Calcein flux was measured as described in Materials and Methods. Relative fluorescence units (F) (mean ± standard deviation) are shown. Panel A shows the rate of accumulation of calcein in MDR1 + CEM/VBL100 (broken line) and wild-type CEM/WTB (solid line) cells. The effects of reserpine on the calcein flux in CEM/VBL100 and CEM/WTB cells are shown in panels B and C, respectively. Calcein fluorescence of CEM/VBL100 cells was significantly enhanced when incubated with the MDR1 antagonist reserpine (panel B, solid line) compared to CEM/VBL100 cells incubated with saline (panel B, broken line). By contrast, the calcein fluorescence of wild-type CEM/WTB cells was not different when incubated in the presence of either reserpine (hatched line) or saline (solid line). The P values indicate the statistical differences between the slopes of calcein fluorescence in CEM/WTB and in CEM/VBL100 cells (A) or between cultures incubated with reserpine and controls (B and C).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Human Immunodeficiency Virus Protease Inhibitors Serve as Substrates for Multidrug Transporter Proteins MDR1 and MRP1 but Retain Antiviral Efficacy in Cell Lines Expressing These Transporters

    doi:

    Figure Lengend Snippet: Calcein-AM flux in multidrug-resistant CEM cells. Calcein flux was measured as described in Materials and Methods. Relative fluorescence units (F) (mean ± standard deviation) are shown. Panel A shows the rate of accumulation of calcein in MDR1 + CEM/VBL100 (broken line) and wild-type CEM/WTB (solid line) cells. The effects of reserpine on the calcein flux in CEM/VBL100 and CEM/WTB cells are shown in panels B and C, respectively. Calcein fluorescence of CEM/VBL100 cells was significantly enhanced when incubated with the MDR1 antagonist reserpine (panel B, solid line) compared to CEM/VBL100 cells incubated with saline (panel B, broken line). By contrast, the calcein fluorescence of wild-type CEM/WTB cells was not different when incubated in the presence of either reserpine (hatched line) or saline (solid line). The P values indicate the statistical differences between the slopes of calcein fluorescence in CEM/WTB and in CEM/VBL100 cells (A) or between cultures incubated with reserpine and controls (B and C).

    Article Snippet: After a 2-min incubation, calcein-AM was added to a final concentration of 1 μM and the plates were placed in a cytofluorimeter (Millipore, Bedford, Mass.).

    Techniques: Fluorescence, Standard Deviation, Incubation

    Effects of various HIV PIs on calcein accumulation in MDR1 + CEM/VBL100 cells. The rates of accumulation of calcein in CEM/VBL100 cells incubated with 5 μM (each) saquinavir (A), ritonavir (B), indinavir (C), or nelfinavir (D) are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence in cultures incubated with HIV PIs and in controls. A statistically significant increase in calcein fluorescence was seen in the presence of ritonavir, nelfinavir, and indinavir, suggesting that they all inhibit MDR1. Significant differences were also observed with saquinavir, albeit at higher (50 μM) concentrations (data not shown).

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Human Immunodeficiency Virus Protease Inhibitors Serve as Substrates for Multidrug Transporter Proteins MDR1 and MRP1 but Retain Antiviral Efficacy in Cell Lines Expressing These Transporters

    doi:

    Figure Lengend Snippet: Effects of various HIV PIs on calcein accumulation in MDR1 + CEM/VBL100 cells. The rates of accumulation of calcein in CEM/VBL100 cells incubated with 5 μM (each) saquinavir (A), ritonavir (B), indinavir (C), or nelfinavir (D) are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence in cultures incubated with HIV PIs and in controls. A statistically significant increase in calcein fluorescence was seen in the presence of ritonavir, nelfinavir, and indinavir, suggesting that they all inhibit MDR1. Significant differences were also observed with saquinavir, albeit at higher (50 μM) concentrations (data not shown).

    Article Snippet: After a 2-min incubation, calcein-AM was added to a final concentration of 1 μM and the plates were placed in a cytofluorimeter (Millipore, Bedford, Mass.).

    Techniques: Incubation, Fluorescence

    Effects of various HIV PIs on calcein accumulation in MDR1 + NB1643Dox r cells. The rates of accumulation of calcein in NB1643Dox r cells incubated with 5 μM (each) ritonavir, nelfinavir, indinavir, or saquinavir are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence (F) in cultures incubated with HIV PIs and in controls.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Human Immunodeficiency Virus Protease Inhibitors Serve as Substrates for Multidrug Transporter Proteins MDR1 and MRP1 but Retain Antiviral Efficacy in Cell Lines Expressing These Transporters

    doi:

    Figure Lengend Snippet: Effects of various HIV PIs on calcein accumulation in MDR1 + NB1643Dox r cells. The rates of accumulation of calcein in NB1643Dox r cells incubated with 5 μM (each) ritonavir, nelfinavir, indinavir, or saquinavir are shown. The broken lines show results of control cultures incubated with saline, while the solid lines show results of cultures incubated with HIV PI. The P values indicate the statistical differences between the slopes of calcein fluorescence (F) in cultures incubated with HIV PIs and in controls.

    Article Snippet: After a 2-min incubation, calcein-AM was added to a final concentration of 1 μM and the plates were placed in a cytofluorimeter (Millipore, Bedford, Mass.).

    Techniques: Incubation, Fluorescence

    Effect of IL-12 on angiogenesis after ICH. ( A ) Flow cytometry analysis of CD45-CD31+ positive cells ratio in hematoma border zone at 7 days after ICH, 8 rats per group; ( B ) CD31 protein expression in hematoma border zone (upper) at 7 days after ICH and gray analysis of protein bands (lower), 8 rats per group; ( C ) Calcein staining was used to detect the tubule formation of BMVES in tubule formation experiment, 3 independent experiments; ( D ) RT-qPCR analysis of angiogenesis-related regulatory gene expression in BMDM after rmIL-12 treatment, 3 independent experiments.

    Journal: Neuropsychiatric Disease and Treatment

    Article Title: Intracerebral Hemorrhage Induced Brain Injury Is Mediated by the Interleukin-12 Receptor in Rats

    doi: 10.2147/NDT.S228773

    Figure Lengend Snippet: Effect of IL-12 on angiogenesis after ICH. ( A ) Flow cytometry analysis of CD45-CD31+ positive cells ratio in hematoma border zone at 7 days after ICH, 8 rats per group; ( B ) CD31 protein expression in hematoma border zone (upper) at 7 days after ICH and gray analysis of protein bands (lower), 8 rats per group; ( C ) Calcein staining was used to detect the tubule formation of BMVES in tubule formation experiment, 3 independent experiments; ( D ) RT-qPCR analysis of angiogenesis-related regulatory gene expression in BMDM after rmIL-12 treatment, 3 independent experiments.

    Article Snippet: 5 μM of calcein (17783, sigma, USA) was added to the wells, and incubated at 37 °C for 30 mins, and then washed 3 times with PBS.

    Techniques: Flow Cytometry, Expressing, Staining, Quantitative RT-PCR

    Effect of IL-12 blockade on M2 macrophage activation and JAK2/STAT4 pathway in hematoma border zone of ICH rats. ( A ) Flow cytometry analysis of iNOS+ (a) and Arginase1+ (b) cells in CD45+Gr-1-CD11b+ macrophages in hematoma border zone of ICH rats and ICH rats treated with anti-IL-12 at 7 days after ICH, 7 rats per group; ( B ) JAK2, p-JAK2, STAT4 and p-STAT4 protein expression in hematoma border zone of ICH rats and ICH rats treated with anti-IL-12 at 7 days after ICH, 7 rats per group; ( C ) JAK2, p-JAK2, STAT4 and p-STAT4 protein expression in BMDM after rmIL-12 and Rap treatment, 3 independent experiments; ( D ) Calcein staining was used to detect the tubule formation of BMVES in tubule formation experiment, 3 independent experiments.

    Journal: Neuropsychiatric Disease and Treatment

    Article Title: Intracerebral Hemorrhage Induced Brain Injury Is Mediated by the Interleukin-12 Receptor in Rats

    doi: 10.2147/NDT.S228773

    Figure Lengend Snippet: Effect of IL-12 blockade on M2 macrophage activation and JAK2/STAT4 pathway in hematoma border zone of ICH rats. ( A ) Flow cytometry analysis of iNOS+ (a) and Arginase1+ (b) cells in CD45+Gr-1-CD11b+ macrophages in hematoma border zone of ICH rats and ICH rats treated with anti-IL-12 at 7 days after ICH, 7 rats per group; ( B ) JAK2, p-JAK2, STAT4 and p-STAT4 protein expression in hematoma border zone of ICH rats and ICH rats treated with anti-IL-12 at 7 days after ICH, 7 rats per group; ( C ) JAK2, p-JAK2, STAT4 and p-STAT4 protein expression in BMDM after rmIL-12 and Rap treatment, 3 independent experiments; ( D ) Calcein staining was used to detect the tubule formation of BMVES in tubule formation experiment, 3 independent experiments.

    Article Snippet: 5 μM of calcein (17783, sigma, USA) was added to the wells, and incubated at 37 °C for 30 mins, and then washed 3 times with PBS.

    Techniques: Activation Assay, Flow Cytometry, Expressing, Staining

    DNA damage and cell death in CuONPs-treated HUVECs. ( A ) MTS assay of HUVECs treated with different concentrations of CuONPs (0, 10, 20, or 40 μg/mL) for 24 h. ( B ) Representative FACS data for CuONPs-treated HUVECs after Calcein AM (live cell fluorescent probe) staining. ( C ) Comet assay to evaluate CuONPs-induced DNA damage in HUVECs. ( D ) Immunofluorescence assay of γH2AX foci formation in CuONPs-treated HUVECs. ( E ) Western blotting assay of phospho-ATR, phospho-ATM, phospho-p53 and phospho-H2AX (γH2AX) in HUVECs treated with CuONPs (20 μg/mL) for 0, 1, 3, 6 and 12 h. Actin was used as a loading control. In ( A ), one-way ANOVA followed by Tukey’s test was performed for statistical analysis. In ( B ), unpaired Student’s t -tests were performed for statistical analysis. ** p

    Journal: International Journal of Nanomedicine

    Article Title: Copper Oxide Nanoparticles Induce Oxidative DNA Damage and Cell Death via Copper Ion-Mediated P38 MAPK Activation in Vascular Endothelial Cells

    doi: 10.2147/IJN.S241157

    Figure Lengend Snippet: DNA damage and cell death in CuONPs-treated HUVECs. ( A ) MTS assay of HUVECs treated with different concentrations of CuONPs (0, 10, 20, or 40 μg/mL) for 24 h. ( B ) Representative FACS data for CuONPs-treated HUVECs after Calcein AM (live cell fluorescent probe) staining. ( C ) Comet assay to evaluate CuONPs-induced DNA damage in HUVECs. ( D ) Immunofluorescence assay of γH2AX foci formation in CuONPs-treated HUVECs. ( E ) Western blotting assay of phospho-ATR, phospho-ATM, phospho-p53 and phospho-H2AX (γH2AX) in HUVECs treated with CuONPs (20 μg/mL) for 0, 1, 3, 6 and 12 h. Actin was used as a loading control. In ( A ), one-way ANOVA followed by Tukey’s test was performed for statistical analysis. In ( B ), unpaired Student’s t -tests were performed for statistical analysis. ** p

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) AssayFACS assays were performed as previously described., , Calcein AM (50 nM, Thermo Fisher Scientific) was used to determine cell viability.

    Techniques: MTS Assay, FACS, Staining, Single Cell Gel Electrophoresis, Immunofluorescence, Western Blot

    Copper ions chelator TTM alleviated HUVECs DNA damage and cell death induced by CuONPs. ( A ) and ( B ) Representative FACS data regarding cellular superoxide anions and mitochondrial superoxide anions after staining with the fluorescent probe DHE and MitoSOX, respectively. HUVECs were pretreated with different concentrations of the copper ions chelator TTM (0, 0.03 and 0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. ( C ) Lipid peroxidation (MDA) assay of HUVECs pretreated with or without TTM (0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. ( D ) Western blotting assay of phosphor-p38, phosphor-Hsp27 and phosphor-ATF-2 in HUVECs, pretreated with or without TTM (0, 0.03, and 0.1mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. Actin was used as a loading control. ( E ) Immunofluorescence assay of the level of γH2AX in CuONPs-treated HUVECs, pretreated with or without TTM (0.1 mM). ( F ) Western blotting assay of phospho-ATR, phospho-ATM, phospho-p53 and γH2AX in HUVECs, which were pretreated with or without TTM (0, 0.03, and 0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. Actin was used as a loading control. ( G ) MTS assay of HUVECs viability after pretreatment with different concentrations of the copper ions chelator TTM (0, 0.03, and 0.1 mM) for 1 h and then treatment with CuONPs (20 or 40 μg/mL) for 24 h. ( H ) Representative FACS data for Calcein AM fluorescence in HUVECs, which were pretreated with or without TTM (0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. In ( C ) and ( G ), one-way ANOVA followed by Tukey’s test was performed for statistical analysis. ** p

    Journal: International Journal of Nanomedicine

    Article Title: Copper Oxide Nanoparticles Induce Oxidative DNA Damage and Cell Death via Copper Ion-Mediated P38 MAPK Activation in Vascular Endothelial Cells

    doi: 10.2147/IJN.S241157

    Figure Lengend Snippet: Copper ions chelator TTM alleviated HUVECs DNA damage and cell death induced by CuONPs. ( A ) and ( B ) Representative FACS data regarding cellular superoxide anions and mitochondrial superoxide anions after staining with the fluorescent probe DHE and MitoSOX, respectively. HUVECs were pretreated with different concentrations of the copper ions chelator TTM (0, 0.03 and 0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. ( C ) Lipid peroxidation (MDA) assay of HUVECs pretreated with or without TTM (0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. ( D ) Western blotting assay of phosphor-p38, phosphor-Hsp27 and phosphor-ATF-2 in HUVECs, pretreated with or without TTM (0, 0.03, and 0.1mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. Actin was used as a loading control. ( E ) Immunofluorescence assay of the level of γH2AX in CuONPs-treated HUVECs, pretreated with or without TTM (0.1 mM). ( F ) Western blotting assay of phospho-ATR, phospho-ATM, phospho-p53 and γH2AX in HUVECs, which were pretreated with or without TTM (0, 0.03, and 0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. Actin was used as a loading control. ( G ) MTS assay of HUVECs viability after pretreatment with different concentrations of the copper ions chelator TTM (0, 0.03, and 0.1 mM) for 1 h and then treatment with CuONPs (20 or 40 μg/mL) for 24 h. ( H ) Representative FACS data for Calcein AM fluorescence in HUVECs, which were pretreated with or without TTM (0.1 mM) for 1 h and then treated with CuONPs (20 μg/mL) for 12 h. In ( C ) and ( G ), one-way ANOVA followed by Tukey’s test was performed for statistical analysis. ** p

    Article Snippet: Fluorescence Activated Cell Sorting (FACS) AssayFACS assays were performed as previously described., , Calcein AM (50 nM, Thermo Fisher Scientific) was used to determine cell viability.

    Techniques: FACS, Staining, Multiple Displacement Amplification, Western Blot, Immunofluorescence, MTS Assay, Fluorescence

    Mdivi-1 protects neurons from excitotoxicity. (A,B) Neurons were stimulated with (A) NMDA (30 μM and 100 μM, 30 min) or (B) AMPA (25 μM, 30 min) and (CTZ, 100 μM) in the presence or absence of mdivi-1 (50 μM, 1 h) and 24 h later cell viability was assessed by the quantification of vital dye calcein-AM fluorescence ( n = 6 and n = 8, respectively). (C) Neurons were exposed to mdivi-1 (50 μM) after being stimulated with 30 and 100 μM of NMDA for 30 min, and 24 h later cell viability was assessed by the analysis of calcein-acetoxymethyl (AM) fluorescence ( n = 3). (D) Neurons were stimulated with 30 μM and 100 μM of NMDA for 30 min in the presence or absence of mdivi-1 (50 μM, 1 h) and 1 h later LDH release to the extracellular medium was quantified ( n = 4). Data represent means ± SEM of normalized calcein fluorescence values. * p

    Journal: Frontiers in Molecular Neuroscience

    Article Title: Mitochondrial Division Inhibitor 1 (mdivi-1) Protects Neurons against Excitotoxicity through the Modulation of Mitochondrial Function and Intracellular Ca2+ Signaling

    doi: 10.3389/fnmol.2018.00003

    Figure Lengend Snippet: Mdivi-1 protects neurons from excitotoxicity. (A,B) Neurons were stimulated with (A) NMDA (30 μM and 100 μM, 30 min) or (B) AMPA (25 μM, 30 min) and (CTZ, 100 μM) in the presence or absence of mdivi-1 (50 μM, 1 h) and 24 h later cell viability was assessed by the quantification of vital dye calcein-AM fluorescence ( n = 6 and n = 8, respectively). (C) Neurons were exposed to mdivi-1 (50 μM) after being stimulated with 30 and 100 μM of NMDA for 30 min, and 24 h later cell viability was assessed by the analysis of calcein-acetoxymethyl (AM) fluorescence ( n = 3). (D) Neurons were stimulated with 30 μM and 100 μM of NMDA for 30 min in the presence or absence of mdivi-1 (50 μM, 1 h) and 1 h later LDH release to the extracellular medium was quantified ( n = 4). Data represent means ± SEM of normalized calcein fluorescence values. * p

    Article Snippet: Reagents and Plasmids Neurobasal® medium, B-27 supplement, antibiotic-antimycotic, calcein acetoxymethyl ester (calcein-AM), JC-1 and rhodamine 123 were purchased from Invitrogen (Barcelona, Spain).

    Techniques: Fluorescence

    GC4403, T2E, and BuOE provide dose-dependent protection of chondrocytes 24 h after 2 J impacts. ( A ) Previously described [ 21 ] increases in DHE staining of chondrocytes within 10× objective confocal images of impacted sites were reproduced and this increase was blunted with treatment by all three mimetics ( n = 3). ( B ) SOD activity in unloaded primary chondrocytes shows a dose responsive increase with all three SOD mimetics ( n = 3). ( C ) Mitochondrial staining with MitoTracker Deep Red following mimetic treatments in the absence of loading shows minimal changes in mitochondrial staining with the exception of the 1000 nM dose of BuOE, though many other mimetic treated samples insignificantly trended towards stronger staining than control ( n = 3). ( D ) Cell counts of Calcein Green AM positive cells from 10× objective confocal images of sites within the impact zone ( n = 3) show maximal protection by GC4403 between 500 nM and 1000 nM, protection by T2E at 250 nM, and protection by BuOE at 1000 nM. ( E ) Mean intensities of MitoTracker Deep Red from cells in ( A ) show maximal benefit from GC4403 at 1000 nM, protection by T2E at 250 nM, and protection by BuOE peaking at 1000 nM ( n = 3). * indicate p

    Journal: Antioxidants

    Article Title: Differential Effects of Superoxide Dismutase Mimetics after Mechanical Overload of Articular Cartilage

    doi: 10.3390/antiox6040098

    Figure Lengend Snippet: GC4403, T2E, and BuOE provide dose-dependent protection of chondrocytes 24 h after 2 J impacts. ( A ) Previously described [ 21 ] increases in DHE staining of chondrocytes within 10× objective confocal images of impacted sites were reproduced and this increase was blunted with treatment by all three mimetics ( n = 3). ( B ) SOD activity in unloaded primary chondrocytes shows a dose responsive increase with all three SOD mimetics ( n = 3). ( C ) Mitochondrial staining with MitoTracker Deep Red following mimetic treatments in the absence of loading shows minimal changes in mitochondrial staining with the exception of the 1000 nM dose of BuOE, though many other mimetic treated samples insignificantly trended towards stronger staining than control ( n = 3). ( D ) Cell counts of Calcein Green AM positive cells from 10× objective confocal images of sites within the impact zone ( n = 3) show maximal protection by GC4403 between 500 nM and 1000 nM, protection by T2E at 250 nM, and protection by BuOE at 1000 nM. ( E ) Mean intensities of MitoTracker Deep Red from cells in ( A ) show maximal benefit from GC4403 at 1000 nM, protection by T2E at 250 nM, and protection by BuOE peaking at 1000 nM ( n = 3). * indicate p

    Article Snippet: Cell viability was confirmed by staining for 30 min with 1 mg/mL Calcein Green AM (Life Technologies, Waltham, MA, USA) for viable cells.

    Techniques: Staining, Activity Assay

    All keratocytes participate in sphere formation. A : Freshly isolated primary keratocytes were cultured in polyHEMA-coated dishes either in Advanced-DMEM (ADV, dark solid bars) or in Advanced-DMEM containing transforming growth factor beta-1 (TGFβ-1), fibroblast growth factor 2 (FGF2), and platelet derived growth factor BB (PDGF; light-stippled bars). Viable cells were labeled with Calcein AM, and the number labeled cells was determined by direct counting after trypsinization. Cell number is shown as a percentage of viable cells added to the original culture. B : Primary stromal cells were plated in Advanced-DMEM containing 10 ng/ml FGF2 under attachment conditions (solid lines) or as spheroids in polyHEMA-coated dishes (broken lines). Cells numbers were estimated by conversion of the dye Alamar Blue to a fluorescent form and normalized to the number of cells added to the culture. Error bars show the SD of triplicate analyses.

    Journal: Molecular Vision

    Article Title: Keratocyte phenotype is enhanced in the absence of attachment to the substratum

    doi:

    Figure Lengend Snippet: All keratocytes participate in sphere formation. A : Freshly isolated primary keratocytes were cultured in polyHEMA-coated dishes either in Advanced-DMEM (ADV, dark solid bars) or in Advanced-DMEM containing transforming growth factor beta-1 (TGFβ-1), fibroblast growth factor 2 (FGF2), and platelet derived growth factor BB (PDGF; light-stippled bars). Viable cells were labeled with Calcein AM, and the number labeled cells was determined by direct counting after trypsinization. Cell number is shown as a percentage of viable cells added to the original culture. B : Primary stromal cells were plated in Advanced-DMEM containing 10 ng/ml FGF2 under attachment conditions (solid lines) or as spheroids in polyHEMA-coated dishes (broken lines). Cells numbers were estimated by conversion of the dye Alamar Blue to a fluorescent form and normalized to the number of cells added to the culture. Error bars show the SD of triplicate analyses.

    Article Snippet: Live-dead cell counting Viable cells were labeled with the vital dye Calcein AM (Invitrogen) at 50 µg/ml for 15 min at 37° in culture medium.

    Techniques: Isolation, Cell Culture, Derivative Assay, Labeling

    Spheroid formation by primary keratocytes. Freshly isolated primary bovine keratocytes were cultured in serum-free conditions on standard tissue culture plastic ( A ) or in vessels coated with polyHEMA, which prevented cell attachment ( B , C , D ). In two days ( B ), cells had formed aggregates, which condensed into smooth spheroids after two weeks of culture ( C ). D : In this panel, two-week spheroids were stained with vital dye Calcein AM to detect live cells (green) and propidium iodide (red) to detect dead cells. Scale bars show 50 µm.

    Journal: Molecular Vision

    Article Title: Keratocyte phenotype is enhanced in the absence of attachment to the substratum

    doi:

    Figure Lengend Snippet: Spheroid formation by primary keratocytes. Freshly isolated primary bovine keratocytes were cultured in serum-free conditions on standard tissue culture plastic ( A ) or in vessels coated with polyHEMA, which prevented cell attachment ( B , C , D ). In two days ( B ), cells had formed aggregates, which condensed into smooth spheroids after two weeks of culture ( C ). D : In this panel, two-week spheroids were stained with vital dye Calcein AM to detect live cells (green) and propidium iodide (red) to detect dead cells. Scale bars show 50 µm.

    Article Snippet: Live-dead cell counting Viable cells were labeled with the vital dye Calcein AM (Invitrogen) at 50 µg/ml for 15 min at 37° in culture medium.

    Techniques: Isolation, Cell Culture, Cell Attachment Assay, Staining

    Fluorescence recovery after photobleaching (FRAP). a Upper panel: Representative diaphragmatic myoblasts transduced with connexin-43 stained with calcein-red in culture for FRAP experiment. Lower panel: images show over time color recovery of the selected cell (arrow) after photobleaching. b Color recovery curves (mean ± SEM at different time points) for diaphragmatic myoblasts and diaphragmatic myoblasts transduced with connexin-43. Higher recovery of intensity in diaphragmatic myoblasts transduced with connexin-43 at each time point can be observed (* p

    Journal: Cytotechnology

    Article Title: Aligned ovine diaphragmatic myoblasts overexpressing human connexin-43 seeded on poly (l-lactic acid) scaffolds for potential use in cardiac regeneration

    doi: 10.1007/s10616-017-0166-4

    Figure Lengend Snippet: Fluorescence recovery after photobleaching (FRAP). a Upper panel: Representative diaphragmatic myoblasts transduced with connexin-43 stained with calcein-red in culture for FRAP experiment. Lower panel: images show over time color recovery of the selected cell (arrow) after photobleaching. b Color recovery curves (mean ± SEM at different time points) for diaphragmatic myoblasts and diaphragmatic myoblasts transduced with connexin-43. Higher recovery of intensity in diaphragmatic myoblasts transduced with connexin-43 at each time point can be observed (* p

    Article Snippet: Cells were stained with 5 µg/ml Calcein Red–Orange AM (ThermoFisher, cat: ) for 30 min at 37 °C, and then washed with HEPES buffer (133 mM NaCl, 5 mM KCl, 1.2 mM MgSO4 , 0.8 mM MgCl2 , 10 mM glucose, 1.35 mM CaCl2 and 10 mM HEPES; pH = 7.35).

    Techniques: Fluorescence, Transduction, Staining

    Effect of IGF-1 on human brain microvascular endothelial cell (hBMECs) survival: A . LDH levels in the media from normoxic cultures were significantly lower than cultures exposed to OGD. IGF-1 treatment, with or without its receptor inhibitor (JB-1) or signaling pathway inhibitors had no effect on OGD-induced LDH levels. B . Calcein assay: Cell survival as measured by calcein was significantly lower in cultures exposed to OGD as compared to normoxic cultures. Here also, IGF-1 treatment, with or without signaling pathway inhibitors showed no significant difference in cell viability. Histograms depict mean and SEM. Each data point is the average of 6 technical replicates, and shown here is the average from 3 separate experiments. *p

    Journal: Experimental neurology

    Article Title: Insulin-Like Growth Factor (IGF)-1 Treatment Stabilizes the Microvascular Cytoskeleton under Ischemic Conditions

    doi: 10.1016/j.expneurol.2018.09.016

    Figure Lengend Snippet: Effect of IGF-1 on human brain microvascular endothelial cell (hBMECs) survival: A . LDH levels in the media from normoxic cultures were significantly lower than cultures exposed to OGD. IGF-1 treatment, with or without its receptor inhibitor (JB-1) or signaling pathway inhibitors had no effect on OGD-induced LDH levels. B . Calcein assay: Cell survival as measured by calcein was significantly lower in cultures exposed to OGD as compared to normoxic cultures. Here also, IGF-1 treatment, with or without signaling pathway inhibitors showed no significant difference in cell viability. Histograms depict mean and SEM. Each data point is the average of 6 technical replicates, and shown here is the average from 3 separate experiments. *p

    Article Snippet: Calcein assay: Cell viability was determined using the Calcein-AM dye (Life Technologies, CA).

    Techniques:

    Immunofluorescence images at 10× ( A , B ) and 63× oil ( C ) magnification using Zeiss Axiovert 200 M Marianas™ Microscope. ( A ) After 14 days of culturing, tissues were enzymatically digested using collagenase. Live separate cells, stained with Calcein AM (green), float as round cells in culture medium. ( B) After 24 hours of additional culturing, the cells were attached to the slide. ( C) Different cell type characteristics and differences in cell nuclei are observed in attached cells. Live cytoplasm is stained with Calcein AM (green) and cell nuclei are stained with DAPI (blue). AM indicates acetoxymethyl and DAPI, 4′,6-diamidino-2-phenylindole.

    Journal: Scientific Reports

    Article Title: An in vitro method to keep human aortic tissue sections functionally and structurally intact

    doi: 10.1038/s41598-018-26549-4

    Figure Lengend Snippet: Immunofluorescence images at 10× ( A , B ) and 63× oil ( C ) magnification using Zeiss Axiovert 200 M Marianas™ Microscope. ( A ) After 14 days of culturing, tissues were enzymatically digested using collagenase. Live separate cells, stained with Calcein AM (green), float as round cells in culture medium. ( B) After 24 hours of additional culturing, the cells were attached to the slide. ( C) Different cell type characteristics and differences in cell nuclei are observed in attached cells. Live cytoplasm is stained with Calcein AM (green) and cell nuclei are stained with DAPI (blue). AM indicates acetoxymethyl and DAPI, 4′,6-diamidino-2-phenylindole.

    Article Snippet: Subsequently, the tissue was incubated in chamber slides for 30 minutes in 300 µl of 0.04% calcein acetoxymethyl (calcein AM) and 0.16% ethidium homodimer-1 (EthD-1) solution (LIVE/DEAD® Viability/Cytotoxicity Kit and PBS solution, Life Technologies, Carlsbad, CA, USA).

    Techniques: Immunofluorescence, Microscopy, Staining

    Analysis of QD655 + -CD11b + and QD585 + -TCR + cell viability (A) and QD655 + -CD11b + adhesion on endothelial monolayers in vitro (B). (A) Cell viability as reported for 20 000 cells. (B) Adhesion of 1 × 10 5 calcein-AM (CAM)-labeled or QD–maurocalcine and CAM-labeled CD11b + cells to bEnd.3 mouse endothelial cell monolayers, as detected by CAM fluorescence intensity. Reported as mean ± S.D, n = 3. * − P

    Journal: Nanotechnology

    Article Title: Quantum dot mediated imaging of atherosclerosis

    doi: 10.1088/0957-4484/20/16/165102

    Figure Lengend Snippet: Analysis of QD655 + -CD11b + and QD585 + -TCR + cell viability (A) and QD655 + -CD11b + adhesion on endothelial monolayers in vitro (B). (A) Cell viability as reported for 20 000 cells. (B) Adhesion of 1 × 10 5 calcein-AM (CAM)-labeled or QD–maurocalcine and CAM-labeled CD11b + cells to bEnd.3 mouse endothelial cell monolayers, as detected by CAM fluorescence intensity. Reported as mean ± S.D, n = 3. * − P

    Article Snippet: In some cases, leukocytes were labeled ex vivo with 1 μ M calcein-AM for 30 min according to the manufacturer's instructions (Invitrogen).

    Techniques: In Vitro, Chick Chorioallantoic Membrane Assay, Labeling, Fluorescence

    Killing of murine lymphoma cells by anti–third-party Tcm is TCR independent and mediated by ICAM-1/LFA-1 interactions and engagement of CD8 molecules. (A) CalceinAM-prelabeled murine A20 lymphoma cells were incubated with or without OT1/Rag −/−

    Journal: Blood

    Article Title: A new approach for eradication of residual lymphoma cells by host nonreactive anti–third-party central memory CD8 T cells

    doi: 10.1182/blood-2012-06-432443

    Figure Lengend Snippet: Killing of murine lymphoma cells by anti–third-party Tcm is TCR independent and mediated by ICAM-1/LFA-1 interactions and engagement of CD8 molecules. (A) CalceinAM-prelabeled murine A20 lymphoma cells were incubated with or without OT1/Rag −/−

    Article Snippet: Lymphoma cells were obtained by Ficoll density gradient centrifugation, after which they were labeled according to the manufacturer’s instructions with 0.15 µg/mL CalceinAM (Invitrogen, Carlsbad, CA), a vital dye that is released upon cell death.

    Techniques: Incubation

    Apoptosis induction in mouse lymphoma cells by anti–third-party Tcm through a nonalloreactive mechanism. CalceinAM-prelabeled murine A20 lymphoma cells (BALB/c origin) were incubated for 16 hours with or without a 5-fold excess of F1 (CB6-H2 bd

    Journal: Blood

    Article Title: A new approach for eradication of residual lymphoma cells by host nonreactive anti–third-party central memory CD8 T cells

    doi: 10.1182/blood-2012-06-432443

    Figure Lengend Snippet: Apoptosis induction in mouse lymphoma cells by anti–third-party Tcm through a nonalloreactive mechanism. CalceinAM-prelabeled murine A20 lymphoma cells (BALB/c origin) were incubated for 16 hours with or without a 5-fold excess of F1 (CB6-H2 bd

    Article Snippet: Lymphoma cells were obtained by Ficoll density gradient centrifugation, after which they were labeled according to the manufacturer’s instructions with 0.15 µg/mL CalceinAM (Invitrogen, Carlsbad, CA), a vital dye that is released upon cell death.

    Techniques: Incubation

    (A) The in vitro cytotoxicity effects of free silibinin, PNs, SPNs, SIPNs, IPNs with NIR irradiation and SIPNs with NIR irradiation at different concentrations on 4T1 cells after 24 h incubation are shown. (B) The photothermal cytotoxicity images of the nanoparticles in 4T1 cells are represented. The live cells were stained with calcein AM (green), and the dead cells were stained with EthD-1 (red). Scale bar=100 μm.

    Journal: Acta Pharmacologica Sinica

    Article Title: Silibinin and indocyanine green-loaded nanoparticles inhibit the growth and metastasis of mammalian breast cancer cells in vitro

    doi: 10.1038/aps.2016.20

    Figure Lengend Snippet: (A) The in vitro cytotoxicity effects of free silibinin, PNs, SPNs, SIPNs, IPNs with NIR irradiation and SIPNs with NIR irradiation at different concentrations on 4T1 cells after 24 h incubation are shown. (B) The photothermal cytotoxicity images of the nanoparticles in 4T1 cells are represented. The live cells were stained with calcein AM (green), and the dead cells were stained with EthD-1 (red). Scale bar=100 μm.

    Article Snippet: Then, the cells treated with IPNs and SIPNs with NIR irradiation were irradiated at 1.5 W·cm−2 for 5 min and incubated for another 12 h. The cells treated with SIPNs were incubated in the dark for another 12 h. Then, the live and dead cells were stained with calcein-AM (AM) and ethidium homodimer-1 (EthD-1) using the LIVE/DEADViability/Cytotoxicity Kit (Life Technologies) and were detected using a fluorescence microscope (IX81, Olympus, Japan).

    Techniques: In Vitro, Irradiation, Incubation, Staining, Ethidium Homodimer Assay

    Influence of RA on follicle viability. (A) Fluorescent staining with calcein-AM/ethidium homodimer-1 after culture in 5 μM retinoic acid for 7 d. Green fluorescence indicates viable follicles/cell. Bar = 100 μm. (B) Mean (± SEM) of follicle viability in freshly collected cat ovarian tissue and cortical pieces cultured for 7 d. Different letters indicate significant (P

    Journal: PLoS ONE

    Article Title: Retinoic acid promotes in vitro follicle activation in the cat ovary by regulating expression of matrix metalloproteinase 9

    doi: 10.1371/journal.pone.0202759

    Figure Lengend Snippet: Influence of RA on follicle viability. (A) Fluorescent staining with calcein-AM/ethidium homodimer-1 after culture in 5 μM retinoic acid for 7 d. Green fluorescence indicates viable follicles/cell. Bar = 100 μm. (B) Mean (± SEM) of follicle viability in freshly collected cat ovarian tissue and cortical pieces cultured for 7 d. Different letters indicate significant (P

    Article Snippet: Follicle viability within the ovarian cortical tissues was evaluated at Day 0 (fresh control; day of tissue excision and initial incubation) or Day 7 of in vitro culture using calcein AM/ethidium homodimer-1 staining (Invitrogen) and observation under a fluorescent microscope (Olympus BX40; Olympus America Inc., Central Valley, PA).

    Techniques: Staining, Fluorescence, Cell Culture

    YC-1 had no influence on RGC-5 cell death. A : After treatment with 20 µM YC-1 or 0.066% DMSO for 24 h, RGC-5 cells were stained with calcein AM (green for live cells), EthD-1 (red for dead cells), and Hoechst 33342 (blue for nuclei) for 40 min at room temperature. Representative photomicrographs show the cell density and composition. B : Cell viability was examined by calcein AM/EthD-1 fluorescence staining and flow cytometry.

    Journal: Molecular Vision

    Article Title: YC-1 targeting of hypoxia-inducible factor-1? reduces RGC-5 cell viability and inhibits cell proliferation

    doi:

    Figure Lengend Snippet: YC-1 had no influence on RGC-5 cell death. A : After treatment with 20 µM YC-1 or 0.066% DMSO for 24 h, RGC-5 cells were stained with calcein AM (green for live cells), EthD-1 (red for dead cells), and Hoechst 33342 (blue for nuclei) for 40 min at room temperature. Representative photomicrographs show the cell density and composition. B : Cell viability was examined by calcein AM/EthD-1 fluorescence staining and flow cytometry.

    Article Snippet: After treatment, cells were incubated with a LIVE/DEAD® Viability/Cytotoxicity Kit (calcein AM/ethidium homodimer-1; Molecular Probes, Eugene, OR) and Hoechst 33342 (Invitrogen Life Technologies) for 30 min at room temperature.

    Techniques: Staining, Ethidium Homodimer Assay, Fluorescence, Flow Cytometry, Cytometry

    The viable chondrocytes within the cell-gel mixture on Calcein-AM/Ethidium homodimer-1 staining : a) A 0-hour and b) A 72-hour culture (×100). The white arrow in b) denotes the dead cells which appear as a reddish color.

    Journal: BMC Musculoskeletal Disorders

    Article Title: Gel-type autologous chondrocyte (Chondron(TM)) implantation for treatment of articular cartilage defects of the knee

    doi: 10.1186/1471-2474-11-103

    Figure Lengend Snippet: The viable chondrocytes within the cell-gel mixture on Calcein-AM/Ethidium homodimer-1 staining : a) A 0-hour and b) A 72-hour culture (×100). The white arrow in b) denotes the dead cells which appear as a reddish color.

    Article Snippet: In addition, the viable status of chondrocytes within the cell-fibrin matrix was grossly examined using Calcein-AM/Ethidium homodimer-1 (Invitrogen, USA) staining.

    Techniques: Staining

    γδ T cells specifically kill Zol-treated HCC cell lines without killing Zol-untreated cells. Bystander cytotoxic activity of γδ T cells was determined in Zol-untreated target cells (HepG2, T2, or PHA blasts). γδ T effector (E) cells (3.0×10 5 cells/well) were co-cultured for 4 h with 5 μM Zol-pretreated (16 h) HepG2 cells (1.0×10 4 cells/well), along with Calcein-AM-labeled, Zol-untreated target (T) cells (1.0×10 4 cells). Zol-pretreated (5 μM, 16 h) HepG2, T2, or PHA blasts served as references. Data represent the mean ± SD of triplicate cultures. Representative results of three independent experiments are shown.

    Journal: International Journal of Oncology

    Article Title: Hepatocellular carcinoma cell sensitivity to Vγ9Vδ2 T lymphocyte-mediated killing is increased by zoledronate

    doi: 10.3892/ijo.2016.3403

    Figure Lengend Snippet: γδ T cells specifically kill Zol-treated HCC cell lines without killing Zol-untreated cells. Bystander cytotoxic activity of γδ T cells was determined in Zol-untreated target cells (HepG2, T2, or PHA blasts). γδ T effector (E) cells (3.0×10 5 cells/well) were co-cultured for 4 h with 5 μM Zol-pretreated (16 h) HepG2 cells (1.0×10 4 cells/well), along with Calcein-AM-labeled, Zol-untreated target (T) cells (1.0×10 4 cells). Zol-pretreated (5 μM, 16 h) HepG2, T2, or PHA blasts served as references. Data represent the mean ± SD of triplicate cultures. Representative results of three independent experiments are shown.

    Article Snippet: Cytotoxicity assay Target cells were labeled with Calcein-AM (Dojindo, Kumamoto, Japan) for 30 min at 37°C and washed three times.

    Techniques: Activity Assay, Cell Culture, Labeling

    In vitro crawling on ICAM-1 or ICAM-2. (A). Neutrophils were fluorescently labelled with Calcein-AM and seeded into un-coated (BSA blocked), or ICAM-2- or ICAM-1-coated chamber slides. Following adhesion, anti-MAC-1 mAb (5 µg/ml) was added to some groups. Images were captured at 2-minute intervals and crawling dynamics were analysed using Imaris. Examples show the position of neutrophils at t = 0 in green, and the crawling tracks over the following 40-minute period in black. Neutrophil crawling speed (B) and total crawling distance (C) was quantified on uncoated, ICAM-2- and ICAM-1-coated glass, with or without anti-MAC-1 mAb. Data were obtained from analysis of > 300 crawling cells per group from four or five mice. The black lines show the mean±s.e.m. for all events analysed. *** P

    Journal: Journal of Cell Science

    Article Title: ICAM-2 facilitates luminal interactions between neutrophils and endothelial cells in vivo

    doi: 10.1242/jcs.137463

    Figure Lengend Snippet: In vitro crawling on ICAM-1 or ICAM-2. (A). Neutrophils were fluorescently labelled with Calcein-AM and seeded into un-coated (BSA blocked), or ICAM-2- or ICAM-1-coated chamber slides. Following adhesion, anti-MAC-1 mAb (5 µg/ml) was added to some groups. Images were captured at 2-minute intervals and crawling dynamics were analysed using Imaris. Examples show the position of neutrophils at t = 0 in green, and the crawling tracks over the following 40-minute period in black. Neutrophil crawling speed (B) and total crawling distance (C) was quantified on uncoated, ICAM-2- and ICAM-1-coated glass, with or without anti-MAC-1 mAb. Data were obtained from analysis of > 300 crawling cells per group from four or five mice. The black lines show the mean±s.e.m. for all events analysed. *** P

    Article Snippet: The neutrophils were fluorescently labelled with Calcein-AM (1 µg/ml, 30 minutes at 37°C), and 50,000 neutrophils per chamber were seeded into culture slides (BD Flacon) and allowed to adhere.

    Techniques: In Vitro, Mouse Assay

    Misoprostol promotes nuclear calcium accumulation and regulates gene expression. (A) H9c2 cells treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. NLS-R-GECO (red) was used to indicate nuclear calcium content in all conditions. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. (B) Quantification of cells in (A), where red fluorescent signal was normalized to cell area and quantified in 10 random fields. (C) H9c2 cells treated as in (A) and Myc-NFATc3 was transfected into each condition. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc primary antibody (green). Cells were then imaged by standard fluorescence microscopy. (D) Quantification of cells in (C), where the number of cells with nuclear Myc-NFATc3, is presented as a percentage of the number of cells/field in 5 random fields. (E) H9c2 cells treated as in (A), Protein extracts were subjected to nuclear/cytosolic fractionation and were immunoblotted, as indicated. (F) H9c2’s treated as in (A). 2 μM CsA was added to half of the conditions for 48 hours in order to inhibit calcineurin-NFAT signalling. Peredox-mCherry (where Red=NAD + , Green=NADH) was used to indicate cytosolic reduced or oxidized NAD content in all conditions. Cells were imaged by standard fluorescence microscopy. (G) Quantification of cells in (F), where the ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) in 15 random fields across 3 independent experiments. (H) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or myc-NFATc3. Peredox-mCherry was used to indicate cytosolic NAD content. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in > 15 random fields across 6 independent experiments. (I) H9c2’s treated as in (H), cells were stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (J) Quantification of Tag-it Violet positive cells in (I), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (K) Immunoblot for MEF2C and MEF2A in protein extracts from PVNCs treated as in (A). (L) PVNCs treated as in (A). RNA was isolated and qRT-PCR was performed for BMP-10 expression. All data are represented as mean ± S.E.M. *P

    Journal: bioRxiv

    Article Title: Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

    doi: 10.1101/681692

    Figure Lengend Snippet: Misoprostol promotes nuclear calcium accumulation and regulates gene expression. (A) H9c2 cells treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. NLS-R-GECO (red) was used to indicate nuclear calcium content in all conditions. Cells were stained with hoechst (blue) and imaged by standard fluorescence microscopy. (B) Quantification of cells in (A), where red fluorescent signal was normalized to cell area and quantified in 10 random fields. (C) H9c2 cells treated as in (A) and Myc-NFATc3 was transfected into each condition. Cells were fixed, stained with hoechst (blue), and immunofluorescence was performed using a Myc primary antibody (green). Cells were then imaged by standard fluorescence microscopy. (D) Quantification of cells in (C), where the number of cells with nuclear Myc-NFATc3, is presented as a percentage of the number of cells/field in 5 random fields. (E) H9c2 cells treated as in (A), Protein extracts were subjected to nuclear/cytosolic fractionation and were immunoblotted, as indicated. (F) H9c2’s treated as in (A). 2 μM CsA was added to half of the conditions for 48 hours in order to inhibit calcineurin-NFAT signalling. Peredox-mCherry (where Red=NAD + , Green=NADH) was used to indicate cytosolic reduced or oxidized NAD content in all conditions. Cells were imaged by standard fluorescence microscopy. (G) Quantification of cells in (F), where the ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) in 15 random fields across 3 independent experiments. (H) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or myc-NFATc3. Peredox-mCherry was used to indicate cytosolic NAD content. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in > 15 random fields across 6 independent experiments. (I) H9c2’s treated as in (H), cells were stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (J) Quantification of Tag-it Violet positive cells in (I), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (K) Immunoblot for MEF2C and MEF2A in protein extracts from PVNCs treated as in (A). (L) PVNCs treated as in (A). RNA was isolated and qRT-PCR was performed for BMP-10 expression. All data are represented as mean ± S.E.M. *P

    Article Snippet: Fluorescent staining, live cell imaging and immunofluorescence Hoechst 3334 and Calcein-AM were all purchased from Biotium and applied using the manufacturer’s protocol.

    Techniques: Expressing, Staining, Fluorescence, Microscopy, Transfection, Immunofluorescence, Fractionation, Isolation, Quantitative RT-PCR

    sNip opposes hypoxia-induced neonatal cardiomyocyte proliferation. (A) Immunoblot for sNip expression in H9c2 protein extracts treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. (B) PVNCs treated with pLenti-HA-sNip ± 10% O 2 (HPX) for 48 hours and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (C) Quantification of Tag-it Violet positive cells in (B), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (D) Quantification of cells in (B), where cell surface area was calculated for > 80 cells/condition in 10 random fields. (E) H9c2 cells treated as in (A), transfected with scrambled control si-RNA or si-sNip, and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (F) Quantification of Tag-it Violet positive cells in (E), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (G) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or HA-sNip. Peredox-mCherry was used to indicate cytosolic NAD content in all conditions. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in > 15 random fields across 3 independent experiments. (H) Ratiometric images of H9c2’s treated as in (G). Laconic was used to indicate cytosolic lactate content. Cells were imaged by FRET-based microscopy. (I) Quantification of cells in (H), where the FRET-YFP (lactate) signal was divided by the YFP (unbound biosensor) signal in 15 random fields across 3 independent experiments. All data are represented as mean ± S.E.M. *P

    Journal: bioRxiv

    Article Title: Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

    doi: 10.1101/681692

    Figure Lengend Snippet: sNip opposes hypoxia-induced neonatal cardiomyocyte proliferation. (A) Immunoblot for sNip expression in H9c2 protein extracts treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. (B) PVNCs treated with pLenti-HA-sNip ± 10% O 2 (HPX) for 48 hours and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (C) Quantification of Tag-it Violet positive cells in (B), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (D) Quantification of cells in (B), where cell surface area was calculated for > 80 cells/condition in 10 random fields. (E) H9c2 cells treated as in (A), transfected with scrambled control si-RNA or si-sNip, and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (F) Quantification of Tag-it Violet positive cells in (E), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (G) Quantification of H9c2 cells treated with 10% O 2 (HPX) for 48 hours and transfected pcDNA3 (control) or HA-sNip. Peredox-mCherry was used to indicate cytosolic NAD content in all conditions. The ratio of green fluorescence (NADH) to red fluorescent signal (NAD + ) was measured in > 15 random fields across 3 independent experiments. (H) Ratiometric images of H9c2’s treated as in (G). Laconic was used to indicate cytosolic lactate content. Cells were imaged by FRET-based microscopy. (I) Quantification of cells in (H), where the FRET-YFP (lactate) signal was divided by the YFP (unbound biosensor) signal in 15 random fields across 3 independent experiments. All data are represented as mean ± S.E.M. *P

    Article Snippet: Fluorescent staining, live cell imaging and immunofluorescence Hoechst 3334 and Calcein-AM were all purchased from Biotium and applied using the manufacturer’s protocol.

    Techniques: Expressing, Staining, Fluorescence, Microscopy, Transfection

    Misoprostol opposes hypoxia-induced neonatal cardiomyocyte proliferation. (A) Immunoblot for Cyclin-D1 and p57(KP39) in protein extracts from primary ventricular neonatal cardiomyocytes (PVNCs) treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. (B) PVNCs treated as in (A) and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (C) Quantification of Tag-it Violet positive cells in (B), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (D) Representative DNA Histograms of H9c2 cells treated as in (B), where G 1 indicates cells that are 2N and G 2 indicates cells that are 4N and G 2 /G 1 ratio indicates the proliferative index. (E) Quantification of cells as in (D), comparing the percentage of cells gated into G 2 vs. G 1 across 6 independent experiments. (F) PVNCs treated as in (B) and stained with calcein-AM (green) and imaged by standard fluorescence microscopy. (G) Quantification of cells in (F), where cell surface area was calculated for > 80 cells/condition in 10 random fields. (H) H9c2’s treated as in (A). 2 mM 2-Deoxy-D-glucose (2DG) was added to half of the conditions for 48 hours in order to inhibit glycolysis. Cells were stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (I) Quantification as in (C) of Tag-it Violet positive cells in (H) for 10 random fields. (J) Quantification of Tag-it Violet positive H9c2 cells treated as in (A), where 100 μM Etomoxir (ETO) was added to half of the conditions for 48 hours in order to inhibit fatty acid oxidation. The number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. All data are represented as mean ± S.E.M. *P

    Journal: bioRxiv

    Article Title: Misoprostol Attenuates Cardiomyocyte Proliferation in the Neonatal Heart through Bnip3 and Perinuclear Calcium Signaling

    doi: 10.1101/681692

    Figure Lengend Snippet: Misoprostol opposes hypoxia-induced neonatal cardiomyocyte proliferation. (A) Immunoblot for Cyclin-D1 and p57(KP39) in protein extracts from primary ventricular neonatal cardiomyocytes (PVNCs) treated with 10 μM misoprostol (Miso) ± 10% O 2 (HPX) for 48 hours. (B) PVNCs treated as in (A) and stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (C) Quantification of Tag-it Violet positive cells in (B), where the number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. (D) Representative DNA Histograms of H9c2 cells treated as in (B), where G 1 indicates cells that are 2N and G 2 indicates cells that are 4N and G 2 /G 1 ratio indicates the proliferative index. (E) Quantification of cells as in (D), comparing the percentage of cells gated into G 2 vs. G 1 across 6 independent experiments. (F) PVNCs treated as in (B) and stained with calcein-AM (green) and imaged by standard fluorescence microscopy. (G) Quantification of cells in (F), where cell surface area was calculated for > 80 cells/condition in 10 random fields. (H) H9c2’s treated as in (A). 2 mM 2-Deoxy-D-glucose (2DG) was added to half of the conditions for 48 hours in order to inhibit glycolysis. Cells were stained with Tag-it Violet (blue) and calcein-AM (green) and imaged by standard fluorescence microscopy. (I) Quantification as in (C) of Tag-it Violet positive cells in (H) for 10 random fields. (J) Quantification of Tag-it Violet positive H9c2 cells treated as in (A), where 100 μM Etomoxir (ETO) was added to half of the conditions for 48 hours in order to inhibit fatty acid oxidation. The number of blue cells is represented as a percentage (%) of the total number of (green) cells in 10 random fields. All data are represented as mean ± S.E.M. *P

    Article Snippet: Fluorescent staining, live cell imaging and immunofluorescence Hoechst 3334 and Calcein-AM were all purchased from Biotium and applied using the manufacturer’s protocol.

    Techniques: Staining, Fluorescence, Microscopy

    TGF-β1 pretreated MSCs display higher adhesivity and traction force after removal of stimulus. (A) TGF-β1 pretreated cells display higher adhesivity on both glass and gel. (B) Pretreated MSCs (CM vs. TGF-β1) plated on glass and PA gels were washed after 3 h to remove detached cells. (Red-Actin, Blue- DAPI, Green- Calcein AM). (C) Traction force heat map of CM and TGF-β1 treated MSCs, (D) TGF-β1 pretreated MSCs exerted higher traction force compared to control after 24 h' removal of stimulus. Values given as mean ± s.e.m.; significance is indicated as * ( p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: TGF-β1 Pretreatment Improves the Function of Mesenchymal Stem Cells in the Wound Bed

    doi: 10.3389/fcell.2017.00028

    Figure Lengend Snippet: TGF-β1 pretreated MSCs display higher adhesivity and traction force after removal of stimulus. (A) TGF-β1 pretreated cells display higher adhesivity on both glass and gel. (B) Pretreated MSCs (CM vs. TGF-β1) plated on glass and PA gels were washed after 3 h to remove detached cells. (Red-Actin, Blue- DAPI, Green- Calcein AM). (C) Traction force heat map of CM and TGF-β1 treated MSCs, (D) TGF-β1 pretreated MSCs exerted higher traction force compared to control after 24 h' removal of stimulus. Values given as mean ± s.e.m.; significance is indicated as * ( p

    Article Snippet: Adhesion assay Control and pretreated MSCs were trypsinized and labeled with a transmembrane fluorescent viability marker, Calcein AM (Anaspec), in HBSS containing divalent ions Ca++ and Mg++.

    Techniques:

    The ndk null-mutant enhances P. aeruginosa virulence in A549 cells through T3SS activation. Prior to cell infection, the indicated bacterial strains were cultured in LB broth for 12 h. The bacterial pellets and culture supernatants were collected after centrifugation. A549 cells were infected with the indicated strains (MOI = 50). In addition, A549 cells were treated with bacterial culture media 5-fold diluted in DMEM. At 2 h post-infection, the cell viability was detected. ( a ) CCK-8 assay. The percentage of cytotoxicity was calculated according to the following formula: cytotoxicity % = 1 − (OD 450 infected cells /OD 450 sham-infected control ) × 100%. ( b ) Calcein-AM staining. Scale bar: 50 μm. Representative images from triplicate experiments are shown. ( c ) Fluorescence intensity per cell from ( b ) was analyzed by Image pro-Plus software (IPP, edition 6.0) (n cell = 90). Data are expressed as the mean ± SD from three independent experiments. *Indicates P

    Journal: Scientific Reports

    Article Title: Ndk, a novel host-responsive regulator, negatively regulates bacterial virulence through quorum sensing in Pseudomonas aeruginosa

    doi: 10.1038/srep28684

    Figure Lengend Snippet: The ndk null-mutant enhances P. aeruginosa virulence in A549 cells through T3SS activation. Prior to cell infection, the indicated bacterial strains were cultured in LB broth for 12 h. The bacterial pellets and culture supernatants were collected after centrifugation. A549 cells were infected with the indicated strains (MOI = 50). In addition, A549 cells were treated with bacterial culture media 5-fold diluted in DMEM. At 2 h post-infection, the cell viability was detected. ( a ) CCK-8 assay. The percentage of cytotoxicity was calculated according to the following formula: cytotoxicity % = 1 − (OD 450 infected cells /OD 450 sham-infected control ) × 100%. ( b ) Calcein-AM staining. Scale bar: 50 μm. Representative images from triplicate experiments are shown. ( c ) Fluorescence intensity per cell from ( b ) was analyzed by Image pro-Plus software (IPP, edition 6.0) (n cell = 90). Data are expressed as the mean ± SD from three independent experiments. *Indicates P

    Article Snippet: In addition, the cell viability was determined by calcein-AM (Santa Cruz) staining .

    Techniques: Mutagenesis, Activation Assay, Infection, Cell Culture, Centrifugation, CCK-8 Assay, Staining, Fluorescence, Software

    Expansion of viable cancer cells is increased in the three-dimensional (3D) cell-sheet model. Calcein-AM (green) and propidium iodide (PI, red) allow observation of viable and dead cells in the cancer spheroid (A) and 3D cell-sheet model (B) with or without CAFs. The spheroid and 3D cell sheet were exposed to vehicle control (ctr), 10 μM sorafenib, or 20 μM cisplatin for 72 h. The area of viable cells was larger in the 3D cell sheet than in the cancer spheroid because of a more peripheral spread of cancer cells in the 3D cell-sheet model. Bars indicate 100 μm.

    Journal: Theranostics

    Article Title: Development of an in vitro cell-sheet cancer model for chemotherapeutic screening

    doi: 10.7150/thno.26439

    Figure Lengend Snippet: Expansion of viable cancer cells is increased in the three-dimensional (3D) cell-sheet model. Calcein-AM (green) and propidium iodide (PI, red) allow observation of viable and dead cells in the cancer spheroid (A) and 3D cell-sheet model (B) with or without CAFs. The spheroid and 3D cell sheet were exposed to vehicle control (ctr), 10 μM sorafenib, or 20 μM cisplatin for 72 h. The area of viable cells was larger in the 3D cell sheet than in the cancer spheroid because of a more peripheral spread of cancer cells in the 3D cell-sheet model. Bars indicate 100 μm.

    Article Snippet: Visualization of cancer cell viability, apoptosis, and hypoxia in the 3D cell-sheet model Figure shows viable and dead cells in cancer spheroids and 3D cell sheets after calcein-AM and PI staining, respectively.

    Techniques:

    Hypoxia observed in the 3D cell-sheet model. (A) Hypoxic cells were detected with LOX-1 staining (red) and live cells with calcein-AM (green). (B) Areas of live and hypoxic cells were measured in the 3D cell sheet with or without CAFs (C alone) at 72 h after exposure to vehicle control, 10 μM sorafenib, or 20 μM cisplatin. The areas of live cells in the cancer-CAFs or treatment groups were compared to the control of C alone and the hypoxic cell areas were relative to the control of C alone or the C:CAF group. Bars indicate 100 μm. The error bars represent the standard deviation from three replicates. * P

    Journal: Theranostics

    Article Title: Development of an in vitro cell-sheet cancer model for chemotherapeutic screening

    doi: 10.7150/thno.26439

    Figure Lengend Snippet: Hypoxia observed in the 3D cell-sheet model. (A) Hypoxic cells were detected with LOX-1 staining (red) and live cells with calcein-AM (green). (B) Areas of live and hypoxic cells were measured in the 3D cell sheet with or without CAFs (C alone) at 72 h after exposure to vehicle control, 10 μM sorafenib, or 20 μM cisplatin. The areas of live cells in the cancer-CAFs or treatment groups were compared to the control of C alone and the hypoxic cell areas were relative to the control of C alone or the C:CAF group. Bars indicate 100 μm. The error bars represent the standard deviation from three replicates. * P

    Article Snippet: Visualization of cancer cell viability, apoptosis, and hypoxia in the 3D cell-sheet model Figure shows viable and dead cells in cancer spheroids and 3D cell sheets after calcein-AM and PI staining, respectively.

    Techniques: Staining, Standard Deviation

    Stable knockdown of ALKBH3 and RAD51C results in severe MMS-induced cytotoxicity in PC3 and NCI-H23 cells. ( A ) HEK293T was transiently transfected with shRNA for 72 h. Knockdown of ALKBH3 and RAD51C was confirmed by western blotting with indicated antibodies. Effect of stable knockdown of ALKBH3 and RAD51C on ( B ) PC-3 and ( C ) NCI-H23. Cells with indicated shRNAs were exposed to various concentrations of MMS (0, 50, 100, 200, 300 and 400 μM) for 48 h; then cell viability was evaluated by MTT assay. Error bar represent the means ± SD of percentage cell viability from four representative experiments. ( D ) Analysis of MMS sensitivity using Calcein-AM assay. Representative images of intracellular Calcein AM esterase activity of PC-3 and ( F ) NCI-H23 cells. Cells were treated with MMS(400uM) for 48h and fluorescent images were captured using an inverted microscope. Bar chart represents the percentage (%) of MMS-induced cytotoxicity of ( E ) PC-3 and ( G ) NCI-H23 cells for the indicated shRNAs.

    Journal: Nucleic Acids Research

    Article Title: Human RAD51 paralogue RAD51C fosters repair of alkylated DNA by interacting with the ALKBH3 demethylase

    doi: 10.1093/nar/gkz938

    Figure Lengend Snippet: Stable knockdown of ALKBH3 and RAD51C results in severe MMS-induced cytotoxicity in PC3 and NCI-H23 cells. ( A ) HEK293T was transiently transfected with shRNA for 72 h. Knockdown of ALKBH3 and RAD51C was confirmed by western blotting with indicated antibodies. Effect of stable knockdown of ALKBH3 and RAD51C on ( B ) PC-3 and ( C ) NCI-H23. Cells with indicated shRNAs were exposed to various concentrations of MMS (0, 50, 100, 200, 300 and 400 μM) for 48 h; then cell viability was evaluated by MTT assay. Error bar represent the means ± SD of percentage cell viability from four representative experiments. ( D ) Analysis of MMS sensitivity using Calcein-AM assay. Representative images of intracellular Calcein AM esterase activity of PC-3 and ( F ) NCI-H23 cells. Cells were treated with MMS(400uM) for 48h and fluorescent images were captured using an inverted microscope. Bar chart represents the percentage (%) of MMS-induced cytotoxicity of ( E ) PC-3 and ( G ) NCI-H23 cells for the indicated shRNAs.

    Article Snippet: Calcein-AM staining After the indicated treatments with MMS as described above, cancer cells were incubated for 30 min at 37°C with 2 μM Calcein AM (C1359, Sigma-Aldrich) diluted in PBS.

    Techniques: Transfection, shRNA, Western Blot, MTT Assay, Calcein AM Assay, Activity Assay, Inverted Microscopy