dispase ii Search Results


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  • 99
    Worthington Biochemical dispase
    Dispase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 971 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dispase/product/Worthington Biochemical
    Average 99 stars, based on 971 article reviews
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    99
    Thermo Fisher dispase
    Dispase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8930 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dispase/product/Thermo Fisher
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    99
    Millipore dispase ii
    Dispase Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1772 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dispase ii/product/Millipore
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    92
    Becton Dickinson dispase
    Neutralization of secreted GDF15 reduces invasion and growth of GDF15-overexpressing ovarian cancer cells. (A) Concentrations of secreted GDF15 were determined by ELISA in media from SKOv3 and Tov21 cell lines. Cells were incubated in serum-free media for 48 h prior to ELISA. Values reflect the average fold expression in three samples per group. (B) Tov21 cells were either untreated, or treated with 1 µg/mL control IgG or GDF15 mAb (147627; R D Systems) for 24 h. The concentration of GDF15 in media was measured by ELISA in triplicates per group. Values reflect the average fold expression per group. (C) Tov21 cells were treated with 1 µg/mL control IgG or GDF15 mAb for 24 h. Real-time PCR was then performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and treated with 1 µg/mL control IgG or GDF15 mAb (147627); media was changed twice a week for 3–4 weeks. Matrigel was dissolved using <t>dispase</t> and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown relative to the control group. (E) Tov21 cells were plated in Boyden chambers and treated with 1 µg/mL control IgG or GDF15 mAb (147627). After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group.
    Dispase, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 92/100, based on 2540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim dispase ii
    Emigrant cells from epidermal, but not dermal, sheets selectively carry macrophage tropic virus. Abraded skin explants were exposed to 10,000 TCID 50 of HIV-1 Ba-L by adding virus to the culture medium. After overnight incubation, the virus was removed, half the treated skin explants were left intact, and the remainder were treated with <t>dispase</t> to enable the epidermis to be separated from the dermis. ( A ) The skin explants ( Split skin ), and epidermal and dermal sheets were cultured by floating on medium in 6-well plates. After 4 d, migrating cells from the individual wells were harvested and added to unstimulated ( PBL ) and stimulated PBLs (10 6 SEB activated blasts/well) in IL-2 supplemented medium. Cells were lysed on day 14 and analyzed for HIV-1 gag sequences by PCR. ( B ) Virus production in cocultures of stimulated PBLs and emigrant cells from epidermis and dermis. HIV-1 Ba-L was added only to epidermal surface ( top ) and dispase treated after the overnight virus pulse. Virus production was measured by RTase in culture supernatants from intact skin explants ( Split skin ), epidermal sheets ( Epid. sheet ), and the dermal sheets ( Dermal sheet ) and plotted against time in culture.
    Dispase Ii, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 254 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Corning Life Sciences dispase
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Dispase, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore collagenase dispase
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Collagenase Dispase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 467 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM dispase
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Dispase, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dispase/product/FUJIFILM
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    99
    Millipore dispase ii solution
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Dispase Ii Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim collagenase dispase
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Collagenase Dispase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore collagenase dispase solution
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Collagenase Dispase Solution, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 70 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Sanko Junyaku Co Ltd dispase ii
    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using <t>dispase</t> to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.
    Dispase Ii, supplied by Sanko Junyaku Co Ltd, used in various techniques. Bioz Stars score: 93/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim dispase solution
    Quantitative measurements of keratinocyte cell adhesion as determined by a novel <t>dispase-based</t> assay. ( A ) Primary keratinocytes under low calcium conditions ( Low Ca ) or treated with calcium for 2 or 9 h ( Ca ) were incubated with dispase for 35 min as described in Materials and Methods. Data are expressed as percentage of single cells released by mechanical disruption after dispase treatment versus total number of cells recovered after subsequent treatment of the same samples with trypsin. ( B ) A similar assay was performed with keratinocytes under low calcium conditions or incubated with calcium for 9 h ( Ca ) in the absence or presence of Genistein (100 μM) ( Gen ), Thyrphostin (100 μM) ( Trph ), or PP1 (1 μg/ml). Cells were preincubated with the inhibitors for 2 h before calcium treatment. ( C ) Keratinocytes under low calcium conditions were infected with a control adenovirus expressing the green fluorescent protein ( Ad-GFP ) or a virus expressing a constitutively active form of c-src ( Ad-src ). 24 h after infection, cells were switched to high calcium medium and incubation was continued for additional 24 h ( Ca ). Cells were analyzed by the dispase assay as before, together with control uninfected cells kept in either low or high calcium medium for 24 h.
    Dispase Solution, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dispase solution/product/Boehringer Mannheim
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    Neutralization of secreted GDF15 reduces invasion and growth of GDF15-overexpressing ovarian cancer cells. (A) Concentrations of secreted GDF15 were determined by ELISA in media from SKOv3 and Tov21 cell lines. Cells were incubated in serum-free media for 48 h prior to ELISA. Values reflect the average fold expression in three samples per group. (B) Tov21 cells were either untreated, or treated with 1 µg/mL control IgG or GDF15 mAb (147627; R D Systems) for 24 h. The concentration of GDF15 in media was measured by ELISA in triplicates per group. Values reflect the average fold expression per group. (C) Tov21 cells were treated with 1 µg/mL control IgG or GDF15 mAb for 24 h. Real-time PCR was then performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and treated with 1 µg/mL control IgG or GDF15 mAb (147627); media was changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown relative to the control group. (E) Tov21 cells were plated in Boyden chambers and treated with 1 µg/mL control IgG or GDF15 mAb (147627). After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group.

    Journal: Biochemical pharmacology

    Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

    doi: 10.1016/j.bcp.2012.10.007

    Figure Lengend Snippet: Neutralization of secreted GDF15 reduces invasion and growth of GDF15-overexpressing ovarian cancer cells. (A) Concentrations of secreted GDF15 were determined by ELISA in media from SKOv3 and Tov21 cell lines. Cells were incubated in serum-free media for 48 h prior to ELISA. Values reflect the average fold expression in three samples per group. (B) Tov21 cells were either untreated, or treated with 1 µg/mL control IgG or GDF15 mAb (147627; R D Systems) for 24 h. The concentration of GDF15 in media was measured by ELISA in triplicates per group. Values reflect the average fold expression per group. (C) Tov21 cells were treated with 1 µg/mL control IgG or GDF15 mAb for 24 h. Real-time PCR was then performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and treated with 1 µg/mL control IgG or GDF15 mAb (147627); media was changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown relative to the control group. (E) Tov21 cells were plated in Boyden chambers and treated with 1 µg/mL control IgG or GDF15 mAb (147627). After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group.

    Article Snippet: Matrigel was digested using dispase (BD Biosciences), and viable cells were counted by trypan blue exclusion.

    Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Incubation, Expressing, Concentration Assay, Real-time Polymerase Chain Reaction

    GDF15 promotes anchorage-independent growth of ovarian cancer cells. (A) CaOv3, OvCar3, and SKOv3 cells were incubated in serum-free media for 48 h. GDF15 concentration was then determined by ELISA in media from cell lines. (B) SKOv3, OvCar3, and CaOv3 ovarian cancer cells were plated in matrigel, and maintained in media containing 20 ng/mL rhGDF15 or vehicle control. Media was changed twice a week for 3–4 weeks. Representative photographs taken with 4× objective lens are shown. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) cell growth is shown for rhGDF15 group (GDF) versus control vehicle group (C) per line. (C) SKOv3 cells were stably transfected with empty vector control or GDF15 expression plasmid. Three GDF15 stable clones (G1, G3, G5) were selected and analyzed by real-time PCR for GDF15 transcript level versus control clone (C). Levels of GDF15 transcript were normalized to RPLPO internal control transcript. Values reflect the average fold change in normalized GDF15 transcript expression per stable clone relative to control clone. (D) Control clone (C) and two GDF15 stable clones (G3 and G5) were plated in matrigel. Media was changed twice a week for 3–4 weeks. Matrigel was then dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) growth is shown for the GDF15 clones relative to control clone. (E) Growth of SKOv3 parental cells treated with vehicle control or 20 ng/mL rhGDF15, or stable control clone or GDF15 clones G1 or G3 was assessed by Real-Time Cell Analysis. Cell index reflects growth over 24 h, and represents the average of triplicate cultures per group.

    Journal: Biochemical pharmacology

    Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

    doi: 10.1016/j.bcp.2012.10.007

    Figure Lengend Snippet: GDF15 promotes anchorage-independent growth of ovarian cancer cells. (A) CaOv3, OvCar3, and SKOv3 cells were incubated in serum-free media for 48 h. GDF15 concentration was then determined by ELISA in media from cell lines. (B) SKOv3, OvCar3, and CaOv3 ovarian cancer cells were plated in matrigel, and maintained in media containing 20 ng/mL rhGDF15 or vehicle control. Media was changed twice a week for 3–4 weeks. Representative photographs taken with 4× objective lens are shown. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) cell growth is shown for rhGDF15 group (GDF) versus control vehicle group (C) per line. (C) SKOv3 cells were stably transfected with empty vector control or GDF15 expression plasmid. Three GDF15 stable clones (G1, G3, G5) were selected and analyzed by real-time PCR for GDF15 transcript level versus control clone (C). Levels of GDF15 transcript were normalized to RPLPO internal control transcript. Values reflect the average fold change in normalized GDF15 transcript expression per stable clone relative to control clone. (D) Control clone (C) and two GDF15 stable clones (G3 and G5) were plated in matrigel. Media was changed twice a week for 3–4 weeks. Matrigel was then dissolved using dispase and cells were counted by trypan blue exclusion. Average fold change in anchorage-independent (AI) growth is shown for the GDF15 clones relative to control clone. (E) Growth of SKOv3 parental cells treated with vehicle control or 20 ng/mL rhGDF15, or stable control clone or GDF15 clones G1 or G3 was assessed by Real-Time Cell Analysis. Cell index reflects growth over 24 h, and represents the average of triplicate cultures per group.

    Article Snippet: Matrigel was digested using dispase (BD Biosciences), and viable cells were counted by trypan blue exclusion.

    Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Clone Assay, Real-time Polymerase Chain Reaction

    PI3K/mTORc1 and MAPK signaling are activated by GDF15 in ovarian cancer cells. (A) SKOv3 cells were serum starved overnight, and then stimulated with 20 ng/mL rhGDF15 for 2 or 5 min, or with the corresponding volume of vehicle control for 5 min. Western blots of total protein lysates were performed at least 3 times for p-Thr180/ Tyr182 p38MAPK, total p38, p-Thr202/Tyr204 p42/p44 Erk1/2, total Erk1/2, p-S473 Akt, and total Akt; representative blots are shown. Quantification is shown as a ratio of phospho-protein to total protein, and is shown relative to vehicle control. (B) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL rhGDF15, or GDF15 plus 1 µM PI3K inhibitor LY294002, 100 nM MEK inhibitor PD0325901, or 10 µM p38MAPK inhibitor SB203580. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the vehicle control group; ** p

    Journal: Biochemical pharmacology

    Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

    doi: 10.1016/j.bcp.2012.10.007

    Figure Lengend Snippet: PI3K/mTORc1 and MAPK signaling are activated by GDF15 in ovarian cancer cells. (A) SKOv3 cells were serum starved overnight, and then stimulated with 20 ng/mL rhGDF15 for 2 or 5 min, or with the corresponding volume of vehicle control for 5 min. Western blots of total protein lysates were performed at least 3 times for p-Thr180/ Tyr182 p38MAPK, total p38, p-Thr202/Tyr204 p42/p44 Erk1/2, total Erk1/2, p-S473 Akt, and total Akt; representative blots are shown. Quantification is shown as a ratio of phospho-protein to total protein, and is shown relative to vehicle control. (B) SKOv3 cells were plated in matrigel. Cells were maintained in media containing vehicle control, 20 ng/mL rhGDF15, or GDF15 plus 1 µM PI3K inhibitor LY294002, 100 nM MEK inhibitor PD0325901, or 10 µM p38MAPK inhibitor SB203580. Media and drugs were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. Fold change in anchorage-independent (AI) growth is shown relative to the vehicle control group; ** p

    Article Snippet: Matrigel was digested using dispase (BD Biosciences), and viable cells were counted by trypan blue exclusion.

    Techniques: Western Blot

    GDF15 knockdown reduces invasion and growth in association with reduced p-4EBP1 in GDF15-overexpressing ovarian cancer cells. (A) Real-time PCR was performed for GDF15 in SKOv3 and Tov21 cells, and normalized to the level of internal control transcript RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (B) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was then performed for GDF15 and RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (C) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and infected with lentiviral control or GDF15 shRNA; media and virus were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown. (E) Tov21 cells were plated in Boyden chambers and infected with control or GDF15 shRNA. After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (F) Tov21 cells were infected with lentiviral control shRNA or GDF15 shRNA for 48 h. Western blots were performed at least twice for phosphorylated and total 4EBP1, Erk1/2, p38, and Akt; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein above each blot.

    Journal: Biochemical pharmacology

    Article Title: Growth differentiation factor 15 stimulates rapamycin-sensitive ovarian cancer cell growth and invasion

    doi: 10.1016/j.bcp.2012.10.007

    Figure Lengend Snippet: GDF15 knockdown reduces invasion and growth in association with reduced p-4EBP1 in GDF15-overexpressing ovarian cancer cells. (A) Real-time PCR was performed for GDF15 in SKOv3 and Tov21 cells, and normalized to the level of internal control transcript RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (B) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was then performed for GDF15 and RPLPO. Values reflect the average fold change in normalized GDF15 transcript. (C) Tov21 cells were infected with lentiviral GDF15 shRNA or control shRNA. Real-time PCR was performed for MMP-2, MMP-9, and VEGF, and normalized to RPLPO. Values reflect the average fold change in normalized transcript. (D) Tov21 cells were plated in matrigel and infected with lentiviral control or GDF15 shRNA; media and virus were changed twice a week for 3–4 weeks. Matrigel was dissolved using dispase and cells were counted by trypan blue exclusion. The percentage of anchorage-independent (AI) growth is shown. (E) Tov21 cells were plated in Boyden chambers and infected with control or GDF15 shRNA. After 24 h, the number of invaded cells was counted in ten different fields per sample. Values reflect the total number of invaded cells in triplicate cultures per group. (F) Tov21 cells were infected with lentiviral control shRNA or GDF15 shRNA for 48 h. Western blots were performed at least twice for phosphorylated and total 4EBP1, Erk1/2, p38, and Akt; representative blots are shown. Quantification is shown as a ratio of phosphorylated to total protein above each blot.

    Article Snippet: Matrigel was digested using dispase (BD Biosciences), and viable cells were counted by trypan blue exclusion.

    Techniques: Real-time Polymerase Chain Reaction, Infection, shRNA, Western Blot

    ZO/afadin KD disrupts barrier function and resistance to external mechanical stress. (A–D) Occludin and Ecad. Junctional occludin changes from continuous in control or afadin single KD to punctate after ZO KD. ZO/afadin KD dramatically disrupts junctional occludin. Ecad still localizes to regions where occludin is lost but no longer concentrates at ZA (D–D″, asterisks; projection, apical 6 µm). (E and F) Barrier function. TER (E) or dextran flux (F) across monolayer. ZO/afadin KD dramatically reduced barrier function, whereas afadin single KD had no effect. Error bars are SDs of three independent experiments. (G–J) Monolayers detached by dispase and mechanically disrupted by pipetting. (G′–J′) 5× zoomed images. ZO KD did not affect tissue integrity. Afadin single KD slightly reduced sheet integrity. ZO/afadin KD dramatically reduced this (I). (K) Model, ZA actomyosin organization in cells with increased contractility. Contractile actomyosin arrays run parallel to BCJs and make end-on contacts at TCJs, generating a “zig-zag” membrane topology at TCJs. (L) Germband extending wild-type or canoe mutant fly embryos—wild-type myosin localizes to planar polarized cables tightly localized to the ZA at anterior-posterior boundaries (arrows). Canoe loss results in myosin broadening.

    Journal: The Journal of Cell Biology

    Article Title: Remodeling the zonula adherens in response to tension and the role of afadin in this response

    doi: 10.1083/jcb.201506115

    Figure Lengend Snippet: ZO/afadin KD disrupts barrier function and resistance to external mechanical stress. (A–D) Occludin and Ecad. Junctional occludin changes from continuous in control or afadin single KD to punctate after ZO KD. ZO/afadin KD dramatically disrupts junctional occludin. Ecad still localizes to regions where occludin is lost but no longer concentrates at ZA (D–D″, asterisks; projection, apical 6 µm). (E and F) Barrier function. TER (E) or dextran flux (F) across monolayer. ZO/afadin KD dramatically reduced barrier function, whereas afadin single KD had no effect. Error bars are SDs of three independent experiments. (G–J) Monolayers detached by dispase and mechanically disrupted by pipetting. (G′–J′) 5× zoomed images. ZO KD did not affect tissue integrity. Afadin single KD slightly reduced sheet integrity. ZO/afadin KD dramatically reduced this (I). (K) Model, ZA actomyosin organization in cells with increased contractility. Contractile actomyosin arrays run parallel to BCJs and make end-on contacts at TCJs, generating a “zig-zag” membrane topology at TCJs. (L) Germband extending wild-type or canoe mutant fly embryos—wild-type myosin localizes to planar polarized cables tightly localized to the ZA at anterior-posterior boundaries (arrows). Canoe loss results in myosin broadening.

    Article Snippet: Epithelial sheet integrity assay and barrier function Cells were grown in six-well plates for 7 d, washed twice with HBSS, and incubated with a 1:1 mixture of dispase solution (BD Biosciences) in HBSS for 2 h at 37°C.

    Techniques: Mutagenesis

    Emigrant cells from epidermal, but not dermal, sheets selectively carry macrophage tropic virus. Abraded skin explants were exposed to 10,000 TCID 50 of HIV-1 Ba-L by adding virus to the culture medium. After overnight incubation, the virus was removed, half the treated skin explants were left intact, and the remainder were treated with dispase to enable the epidermis to be separated from the dermis. ( A ) The skin explants ( Split skin ), and epidermal and dermal sheets were cultured by floating on medium in 6-well plates. After 4 d, migrating cells from the individual wells were harvested and added to unstimulated ( PBL ) and stimulated PBLs (10 6 SEB activated blasts/well) in IL-2 supplemented medium. Cells were lysed on day 14 and analyzed for HIV-1 gag sequences by PCR. ( B ) Virus production in cocultures of stimulated PBLs and emigrant cells from epidermis and dermis. HIV-1 Ba-L was added only to epidermal surface ( top ) and dispase treated after the overnight virus pulse. Virus production was measured by RTase in culture supernatants from intact skin explants ( Split skin ), epidermal sheets ( Epid. sheet ), and the dermal sheets ( Dermal sheet ) and plotted against time in culture.

    Journal: The Journal of Experimental Medicine

    Article Title: HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin

    doi:

    Figure Lengend Snippet: Emigrant cells from epidermal, but not dermal, sheets selectively carry macrophage tropic virus. Abraded skin explants were exposed to 10,000 TCID 50 of HIV-1 Ba-L by adding virus to the culture medium. After overnight incubation, the virus was removed, half the treated skin explants were left intact, and the remainder were treated with dispase to enable the epidermis to be separated from the dermis. ( A ) The skin explants ( Split skin ), and epidermal and dermal sheets were cultured by floating on medium in 6-well plates. After 4 d, migrating cells from the individual wells were harvested and added to unstimulated ( PBL ) and stimulated PBLs (10 6 SEB activated blasts/well) in IL-2 supplemented medium. Cells were lysed on day 14 and analyzed for HIV-1 gag sequences by PCR. ( B ) Virus production in cocultures of stimulated PBLs and emigrant cells from epidermis and dermis. HIV-1 Ba-L was added only to epidermal surface ( top ) and dispase treated after the overnight virus pulse. Virus production was measured by RTase in culture supernatants from intact skin explants ( Split skin ), epidermal sheets ( Epid. sheet ), and the dermal sheets ( Dermal sheet ) and plotted against time in culture.

    Article Snippet: The skin was incubated with dispase II (5–10 mg/ml; Boehringer Mannheim GmbH ) in RPMI 1640 at 4°C with the dermal side facing up.

    Techniques: Incubation, Cell Culture, Polymerase Chain Reaction

    Epidermal, but not dermal, exposure to HIV-1 results in selective carriage of macrophage tropic virus by emigrant cells. ( A ) Culture conditions were as described in Fig. 2 . HIV-1 228 or HIV-1 Ba-L (10,000 TCID 50 ) was added directly to the underlying culture medium ( Medium ) and to the surface ( Epidermis ) of abraded skin explants (3–4 cm 2 ). After 16 h, the virus was removed and the skin placed back in culture for 4 d. Migrating cells were treated with collagenase, washed, and added to 10 6 unstimulated PBLs. On day 10, coculture cells were lysed and analyzed for gag sequences by semiquantitative PCR. Results are for nine replicate skin explants exposed to 228 and 10 exposed to Ba-L via epidermis, and for duplicate cultures exposed via the culture medium. ( B ) Similar culture conditions to A . Abraded skin explants were exposed to 10,000 TCID 50 of HIV-1 Ba-L or HIV-1 228 by adding virus to the culture medium ( Medium ). After 16 h, the explants were washed and half were left as explants and half were treated with dispase to separate epidermal and dermal layers. Skin explants ( Split skin ) and epidermal sheets ( Epid. Sheet ) were placed back in culture. Emigrating cells were harvested on day 4 and cocultured with 10 6 /well unstimulated or SEB (40 ng/ml) -stimulated PBLs. PCR for HLA-DQ and gag sequences were performed on lysates of cocultures on day 10.

    Journal: The Journal of Experimental Medicine

    Article Title: HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin

    doi:

    Figure Lengend Snippet: Epidermal, but not dermal, exposure to HIV-1 results in selective carriage of macrophage tropic virus by emigrant cells. ( A ) Culture conditions were as described in Fig. 2 . HIV-1 228 or HIV-1 Ba-L (10,000 TCID 50 ) was added directly to the underlying culture medium ( Medium ) and to the surface ( Epidermis ) of abraded skin explants (3–4 cm 2 ). After 16 h, the virus was removed and the skin placed back in culture for 4 d. Migrating cells were treated with collagenase, washed, and added to 10 6 unstimulated PBLs. On day 10, coculture cells were lysed and analyzed for gag sequences by semiquantitative PCR. Results are for nine replicate skin explants exposed to 228 and 10 exposed to Ba-L via epidermis, and for duplicate cultures exposed via the culture medium. ( B ) Similar culture conditions to A . Abraded skin explants were exposed to 10,000 TCID 50 of HIV-1 Ba-L or HIV-1 228 by adding virus to the culture medium ( Medium ). After 16 h, the explants were washed and half were left as explants and half were treated with dispase to separate epidermal and dermal layers. Skin explants ( Split skin ) and epidermal sheets ( Epid. Sheet ) were placed back in culture. Emigrating cells were harvested on day 4 and cocultured with 10 6 /well unstimulated or SEB (40 ng/ml) -stimulated PBLs. PCR for HLA-DQ and gag sequences were performed on lysates of cocultures on day 10.

    Article Snippet: The skin was incubated with dispase II (5–10 mg/ml; Boehringer Mannheim GmbH ) in RPMI 1640 at 4°C with the dermal side facing up.

    Techniques: Polymerase Chain Reaction

    Effects of dispase on HIV and target cells. ( A ) CD4 T cells from peripheral blood were cultured for 2 h at 4°C in dispase concentrations between 0.1 and 10 mg/ml. Cells were washed and labeled with CD4 (OKT4) and leu3a. The leu3a, but not OKT4 (CD4), epitope of the CD4 receptor recognized by HIV-1 was removed at concentrations of 1 mg/ml or greater. ( B ) Virus production from SEB (40 ng/ml)- stimulated PBLs was reduced when dispase-treated virus was used to infect the cells. Dispase treatment for 2 h at 4°C at concentrations of dispase > 0.1 mg/ ml removed RTase activity (RT cpm/μl) and p24 (ng/ml) activity from the HIV-1 Ba-L virus stocks.

    Journal: The Journal of Experimental Medicine

    Article Title: HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin

    doi:

    Figure Lengend Snippet: Effects of dispase on HIV and target cells. ( A ) CD4 T cells from peripheral blood were cultured for 2 h at 4°C in dispase concentrations between 0.1 and 10 mg/ml. Cells were washed and labeled with CD4 (OKT4) and leu3a. The leu3a, but not OKT4 (CD4), epitope of the CD4 receptor recognized by HIV-1 was removed at concentrations of 1 mg/ml or greater. ( B ) Virus production from SEB (40 ng/ml)- stimulated PBLs was reduced when dispase-treated virus was used to infect the cells. Dispase treatment for 2 h at 4°C at concentrations of dispase > 0.1 mg/ ml removed RTase activity (RT cpm/μl) and p24 (ng/ml) activity from the HIV-1 Ba-L virus stocks.

    Article Snippet: The skin was incubated with dispase II (5–10 mg/ml; Boehringer Mannheim GmbH ) in RPMI 1640 at 4°C with the dermal side facing up.

    Techniques: Cell Culture, Labeling, Activity Assay

    Dendritic cells in the epidermis select for macrophage tropic virus. Abraded skin explants were pulsed with 1,000 TCID 50 /ml (∼5,000 TCID 50 ) of HIV-1 Ba-L by adding virus to the medium overnight at 37°C. After dispase treatment, epidermal and dermal sheets were floated in medium for 3 d. Migrating cells were treated with collagenase and separated over a step metrizamide gradient, stained with FITC–HLA-DR and PE-CD3, and sorted for DCs using a cell sorter. ( A ) As previously shown ( 17 – 19 ), cells migrating from the dermis contained DCs, T cells, and DC–T cell conjugates, whereas cells from the epidermis contained DCs and DC–T cell conjugates, but few CD3 + cells. ( B ) Sorted cells were analyzed for their ability to transmit infection by diluting into coculture with T cells. Half log 10 dilutions of the sorted emigrant cells (10 4 ) were performed in 96-well plates with SEB-stimulated PBLs (10 5 /well) in medium containing 5% IL-2. Virus replication in cocultures was assessed by lysing cocultures on day 10 and analyzing for gag sequences using PCR. At the highest cell concentration, the PCR reactions contained the equivalent of 2,500 sorted cells. Duplicate dilution series for DCs sorted from epidermal emigrants and DCs, and DC–T cell conjugates from dermal emigrants from Ba-L exposed skin are shown. Dilution series for DCs sorted from epidermis and dermis of NL43 exposed skin are shown in the lower two panels.

    Journal: The Journal of Experimental Medicine

    Article Title: HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin

    doi:

    Figure Lengend Snippet: Dendritic cells in the epidermis select for macrophage tropic virus. Abraded skin explants were pulsed with 1,000 TCID 50 /ml (∼5,000 TCID 50 ) of HIV-1 Ba-L by adding virus to the medium overnight at 37°C. After dispase treatment, epidermal and dermal sheets were floated in medium for 3 d. Migrating cells were treated with collagenase and separated over a step metrizamide gradient, stained with FITC–HLA-DR and PE-CD3, and sorted for DCs using a cell sorter. ( A ) As previously shown ( 17 – 19 ), cells migrating from the dermis contained DCs, T cells, and DC–T cell conjugates, whereas cells from the epidermis contained DCs and DC–T cell conjugates, but few CD3 + cells. ( B ) Sorted cells were analyzed for their ability to transmit infection by diluting into coculture with T cells. Half log 10 dilutions of the sorted emigrant cells (10 4 ) were performed in 96-well plates with SEB-stimulated PBLs (10 5 /well) in medium containing 5% IL-2. Virus replication in cocultures was assessed by lysing cocultures on day 10 and analyzing for gag sequences using PCR. At the highest cell concentration, the PCR reactions contained the equivalent of 2,500 sorted cells. Duplicate dilution series for DCs sorted from epidermal emigrants and DCs, and DC–T cell conjugates from dermal emigrants from Ba-L exposed skin are shown. Dilution series for DCs sorted from epidermis and dermis of NL43 exposed skin are shown in the lower two panels.

    Article Snippet: The skin was incubated with dispase II (5–10 mg/ml; Boehringer Mannheim GmbH ) in RPMI 1640 at 4°C with the dermal side facing up.

    Techniques: Staining, Infection, Polymerase Chain Reaction, Concentration Assay

    Virus dose for infection and phenotype of emigrant cells from skin explants. ( A ) HIV-1 Ba-L was added to the abraded surface of skin ( epidermis ) or to the culture medium ( medium ) at high (10,000 TCID 50 , closed symbols ) or at low (1,000 TCID 50 , open symbols ) concentrations. After overnight exposure, the explants were washed and returned to culture for 3 d. The emigrants were divided equally into triplicate cocultures with allogeneic T cells (see Materials and Methods). Reverse transcriptase activity was measured at 0, 7, and 9 d of culture. Transmission to T cells occurred after addition of 10,000 TCID 50 , but not 1,000 TCID 50 , of HIV-1 to the epidermal surface. Transmission occurred at both virus doses when exposure was via the culture medium. ( B ) Recovery and phenotype of cells from skin explants and dispase-treated skin. Skin explants with and without epidermal abrasion were cultured and the emigrant cells from skin explants and from the separated epidermal and dermal sheets enumerated and phenotype determined by flow cytometry. The areas of the skin explants and epidermal sheets were measured after culture using a 2-mm grid and the data plotted for cell recovery from triplicate cultures of 8–13 cm 2 of skin. Results are representative of three similar experiments.

    Journal: The Journal of Experimental Medicine

    Article Title: HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin

    doi:

    Figure Lengend Snippet: Virus dose for infection and phenotype of emigrant cells from skin explants. ( A ) HIV-1 Ba-L was added to the abraded surface of skin ( epidermis ) or to the culture medium ( medium ) at high (10,000 TCID 50 , closed symbols ) or at low (1,000 TCID 50 , open symbols ) concentrations. After overnight exposure, the explants were washed and returned to culture for 3 d. The emigrants were divided equally into triplicate cocultures with allogeneic T cells (see Materials and Methods). Reverse transcriptase activity was measured at 0, 7, and 9 d of culture. Transmission to T cells occurred after addition of 10,000 TCID 50 , but not 1,000 TCID 50 , of HIV-1 to the epidermal surface. Transmission occurred at both virus doses when exposure was via the culture medium. ( B ) Recovery and phenotype of cells from skin explants and dispase-treated skin. Skin explants with and without epidermal abrasion were cultured and the emigrant cells from skin explants and from the separated epidermal and dermal sheets enumerated and phenotype determined by flow cytometry. The areas of the skin explants and epidermal sheets were measured after culture using a 2-mm grid and the data plotted for cell recovery from triplicate cultures of 8–13 cm 2 of skin. Results are representative of three similar experiments.

    Article Snippet: The skin was incubated with dispase II (5–10 mg/ml; Boehringer Mannheim GmbH ) in RPMI 1640 at 4°C with the dermal side facing up.

    Techniques: Infection, Activity Assay, Transmission Assay, Cell Culture, Flow Cytometry, Cytometry

    Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using dispase to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.

    Journal: Toxicology in vitro : an international journal published in association with BIBRA

    Article Title: Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane

    doi: 10.1016/j.tiv.2017.03.002

    Figure Lengend Snippet: Evaluation of the cytotoxicity induced by MWCNTs on HBMECs grown on plastic or matrigel HBMECs were plated on standard tissue culture plastic or matrigel coated plastic and recovered overnight. HBMECs grown on plastic (A) or Matrigel (B) were treated for 24 h with increasing doses of different types of MWCNTs as indicated. Cells were lysed, pelleted, and the supernatant was analyzed for ATP content as a measure of cell viability using the CellTiter-Glo assay. Three independent biological replicates were performed with at least three technical replicates per treatment group. Endpoint analysis shows the groups that are significantly different (*p≤0.05, **p≤0.01) at the highest dose (ANOVA; post-hoc T-Test). In parallel, lysates of similarly treated cells grown on plastic (C) or Matrigel (D) were collected and probed for cleaved PARP, a protein indicative of apoptosis, via western blot analysis. GAPDH was used as a loading control. Lysates from U87 cells treated with 10 μM doxorubicin was used as a positive control. Cells grown on matrigel were isolated using dispase to degrade the matrigel without affecting HBMEC cell integrity. The graph below the western blot displays average fold change of cleaved PARP protein levels relative to GAPDH loading control for three independent biological replicates. Fold change is displayed as the mean ± standard deviation of each experiment.

    Article Snippet: For HBMECs grown on Matrigel®, cells were isolated by aspirating normal growth media and adding equivalent culture volumes of Dispase (Corning), incubating at 37 °C for 30 min, then washing 2x with ice cold 1xPBS prior to lysis.

    Techniques: Glo Assay, Western Blot, Positive Control, Isolation, Standard Deviation

    Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).

    Journal: Cell reports

    Article Title: Long-Term In Vitro Expansion of Epithelial Stem Cells Enabled by Pharmacological Inhibition of PAK1-ROCK-Myosin II and TGF-β Signaling

    doi: 10.1016/j.celrep.2018.09.072

    Figure Lengend Snippet: Differentiation of HFKs Expanded with the EpiX Medium (A) Addition of 1 mM CaCl 2 to the EpiX medium induced the HFKs to differentiate in 24 hr. (B) Immunofluorescence staining of tight junctions (ZO-1 and Occludin) in HFKs cultured in EpiX plus 1 mM CaCl 2 for 7 days. ZO-1, zonula occludens-1. (C and D) Confluent HFKs in EpiX (C) or EpiX plus 1 mM CaCl 2 (D). Many domes with liquid accumulated underneath occurred in the culture with EpiX plus 1 mM CaCl 2 . (E) HFKs cultured in a T-75 flask in EpiX plus 1.5 mM CaCl 2 for 7 days form an intact epithelium sheet, which was released from the flask after 30-min incubation in dispase at 37°C. (F) HFKs were differentiated at ALI for 14 days and formed a stratified epithelium. (G) EpiX-expanded HFKs were subcutaneously injected into immune-comprised mice. After 5 weeks, the cells formed cysts which resembled epidermis. (H) Most cells in the basal layer were stained positive for the proliferation marker Ki67. (I) The basal and supra-basal layers of the cystic epithelium stained positive for KRT14 (K14).

    Article Snippet: Keratinocytes isolation and expansion Fresh human skin purchased from ZenBio Inc. was cut into small pieces and placed in Dispase solution (Corning) at 4C overnight.

    Techniques: Immunofluorescence, Staining, Cell Culture, Incubation, Injection, Mouse Assay, Marker

    Quantitative measurements of keratinocyte cell adhesion as determined by a novel dispase-based assay. ( A ) Primary keratinocytes under low calcium conditions ( Low Ca ) or treated with calcium for 2 or 9 h ( Ca ) were incubated with dispase for 35 min as described in Materials and Methods. Data are expressed as percentage of single cells released by mechanical disruption after dispase treatment versus total number of cells recovered after subsequent treatment of the same samples with trypsin. ( B ) A similar assay was performed with keratinocytes under low calcium conditions or incubated with calcium for 9 h ( Ca ) in the absence or presence of Genistein (100 μM) ( Gen ), Thyrphostin (100 μM) ( Trph ), or PP1 (1 μg/ml). Cells were preincubated with the inhibitors for 2 h before calcium treatment. ( C ) Keratinocytes under low calcium conditions were infected with a control adenovirus expressing the green fluorescent protein ( Ad-GFP ) or a virus expressing a constitutively active form of c-src ( Ad-src ). 24 h after infection, cells were switched to high calcium medium and incubation was continued for additional 24 h ( Ca ). Cells were analyzed by the dispase assay as before, together with control uninfected cells kept in either low or high calcium medium for 24 h.

    Journal: The Journal of Cell Biology

    Article Title: Tyrosine Phosphorylation and Src Family Kinases Control Keratinocyte Cell-Cell Adhesion

    doi:

    Figure Lengend Snippet: Quantitative measurements of keratinocyte cell adhesion as determined by a novel dispase-based assay. ( A ) Primary keratinocytes under low calcium conditions ( Low Ca ) or treated with calcium for 2 or 9 h ( Ca ) were incubated with dispase for 35 min as described in Materials and Methods. Data are expressed as percentage of single cells released by mechanical disruption after dispase treatment versus total number of cells recovered after subsequent treatment of the same samples with trypsin. ( B ) A similar assay was performed with keratinocytes under low calcium conditions or incubated with calcium for 9 h ( Ca ) in the absence or presence of Genistein (100 μM) ( Gen ), Thyrphostin (100 μM) ( Trph ), or PP1 (1 μg/ml). Cells were preincubated with the inhibitors for 2 h before calcium treatment. ( C ) Keratinocytes under low calcium conditions were infected with a control adenovirus expressing the green fluorescent protein ( Ad-GFP ) or a virus expressing a constitutively active form of c-src ( Ad-src ). 24 h after infection, cells were switched to high calcium medium and incubation was continued for additional 24 h ( Ca ). Cells were analyzed by the dispase assay as before, together with control uninfected cells kept in either low or high calcium medium for 24 h.

    Article Snippet: Dispase Treatment of Mouse Primary Keratinocytes Duplicate 60-mm dishes of confluent keratinocyte cultures in either low or high calcium medium were washed twice in PBS and incubated in 2 ml dispase solution in PBS (from Bacillus polymyxa ; > 2.4 units/ml; Boehringer Mannheim Corp. ) at 34°C.

    Techniques: Incubation, Infection, Expressing

    Decreased strength of calcium-induced cell adhesion as a consequence of tyrosine kinase inhibition or lack of the Fyn kinase. ( A ) Primary keratinocytes under growing conditions were either tested as untreated controls ( Ctr ) or pretreated for 2 h with Genistein. Incubation was then continued for additional 24 h under high calcium conditions. Cultures were examined as such ( left panels ) or after dispase treatment for 5 min ( right panels ). Arrows , the focal areas of cell detachment that occurred in the tyrosine kinase inhibitor-treated cultures. No such areas were evident in control cultures even after prolonged dispase exposure ( > 30 min). Instead, control cells eventually detached from the dish as a confluent sheet. Similar results were observed with keratinocyte cultures switched to high calcium conditions for only 9 h (not shown). ( B ) Primary keratinocytes derived from fyn −/− mice and wild-type littermates were exposed to high calcium concentrations (2 mM) for 9 h. Cultures were examined as such ( left panels ) or after treatment with dispase for 5 min ( right panels ). Arrows , the focal areas of cell detachment that occurred in the fyn −/− cultures already at this time. There were no cells missing in the monolayer of fyn knockout keratinocytes before dispase treatment. As we previously reported ( Calautti et al., 1995 ), the fyn knockout keratinocytes fail to stratify and are larger than normal, which explains the different morphological appearance of these cultures relative to the wild-type controls even before dispase treatment. Similar results were observed in two other independent experiments. Bar, 60 μm.

    Journal: The Journal of Cell Biology

    Article Title: Tyrosine Phosphorylation and Src Family Kinases Control Keratinocyte Cell-Cell Adhesion

    doi:

    Figure Lengend Snippet: Decreased strength of calcium-induced cell adhesion as a consequence of tyrosine kinase inhibition or lack of the Fyn kinase. ( A ) Primary keratinocytes under growing conditions were either tested as untreated controls ( Ctr ) or pretreated for 2 h with Genistein. Incubation was then continued for additional 24 h under high calcium conditions. Cultures were examined as such ( left panels ) or after dispase treatment for 5 min ( right panels ). Arrows , the focal areas of cell detachment that occurred in the tyrosine kinase inhibitor-treated cultures. No such areas were evident in control cultures even after prolonged dispase exposure ( > 30 min). Instead, control cells eventually detached from the dish as a confluent sheet. Similar results were observed with keratinocyte cultures switched to high calcium conditions for only 9 h (not shown). ( B ) Primary keratinocytes derived from fyn −/− mice and wild-type littermates were exposed to high calcium concentrations (2 mM) for 9 h. Cultures were examined as such ( left panels ) or after treatment with dispase for 5 min ( right panels ). Arrows , the focal areas of cell detachment that occurred in the fyn −/− cultures already at this time. There were no cells missing in the monolayer of fyn knockout keratinocytes before dispase treatment. As we previously reported ( Calautti et al., 1995 ), the fyn knockout keratinocytes fail to stratify and are larger than normal, which explains the different morphological appearance of these cultures relative to the wild-type controls even before dispase treatment. Similar results were observed in two other independent experiments. Bar, 60 μm.

    Article Snippet: Dispase Treatment of Mouse Primary Keratinocytes Duplicate 60-mm dishes of confluent keratinocyte cultures in either low or high calcium medium were washed twice in PBS and incubated in 2 ml dispase solution in PBS (from Bacillus polymyxa ; > 2.4 units/ml; Boehringer Mannheim Corp. ) at 34°C.

    Techniques: Inhibition, Incubation, Derivative Assay, Mouse Assay, Knock-Out