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  • 97
    Worthington Biochemical dispase
    Dispase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 97/100, based on 971 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    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
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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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    92
    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: 92/100, based on 485 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Becton Dickinson dispase
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    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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    99
    Millipore collagenase dispase
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    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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    92
    FUJIFILM dispase
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    Dispase, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 74 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dispase ii solution
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    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
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    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
    https://www.bioz.com/result/collagenase dispase/product/Boehringer Mannheim
    Average 92 stars, based on 155 article reviews
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    collagenase dispase - by Bioz Stars, 2021-01
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    Millipore collagenase dispase solution
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    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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    92
    Sanko Junyaku Co Ltd dispase ii
    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with <t>dispase</t> to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.
    Dispase Ii, supplied by Sanko Junyaku Co Ltd, used in various techniques. Bioz Stars score: 92/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    Merck KGaA dispase i
    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 I, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mouse monoclonal Amyloid beta 40 antibody
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    Purified recombinant beta Amyloid 42 protein
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    Purified recombinant beta Amyloid 40 protein
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    Image Search Results


    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

    Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with dispase to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.

    Journal: PeerJ

    Article Title: Insight into the maintenance of odontogenic potential in mouse dental mesenchymal cells based on transcriptomic analysis

    doi: 10.7717/peerj.1684

    Figure Lengend Snippet: Experimental design. Tooth germs from embryonic day 14.5 mice were obtained and digested with dispase to separate the dental mesenchyme from dental epithelium. Freshly isolated dental mesenchymal cells were designated as P0 and cultured in vitro . RNA samples from the P0, the first (P1), and second (P2) passages were collected before they were submitted for RNA-seq using an Illumina Hiseq™ 2000.

    Article Snippet: The lower first molar tooth germs were dissected from the mandibles of E14.5 mice with fine needles and treated with 0.75 mg/ml dispase (Becton, Dickinson and Co., Franklin Lakes, NJ, USA) for 40 min at 37 °C to facilitate separation from dental epithelium.

    Techniques: Mouse Assay, Isolation, Cell Culture, In Vitro, RNA Sequencing Assay

    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