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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 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "HIV-1 Selection by Epidermal Dendritic Cells during Transmission across Human Skin "

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

Journal: The Journal of Experimental Medicine

doi:

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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

Techniques Used: 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.
Figure Legend 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.

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

2) Product Images from "A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels"

Article Title: A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi:

Effect of anti-MDR-1 mAb on the maturation of DC. The levels of MHC II expressed by epidermal DC were analyzed in gated epidermal suspensions by flow cytometry before the onset of culture (Day 0) or after 3 days of culture in the absence of mAb or in the presence of MRK16. Single cell suspensions of the epidermis were prepared by digestion with dispase followed by trypsin. Keratinocytes and other skin cells were excluded from the analysis by setting a gate to include only MHC II + cells.
Figure Legend Snippet: Effect of anti-MDR-1 mAb on the maturation of DC. The levels of MHC II expressed by epidermal DC were analyzed in gated epidermal suspensions by flow cytometry before the onset of culture (Day 0) or after 3 days of culture in the absence of mAb or in the presence of MRK16. Single cell suspensions of the epidermis were prepared by digestion with dispase followed by trypsin. Keratinocytes and other skin cells were excluded from the analysis by setting a gate to include only MHC II + cells.

Techniques Used: Flow Cytometry, Cytometry

Related Articles

Incubation:

Article Title: A physiologic function for p-glycoprotein (MDR-1) during the migration of dendritic cells from skin via afferent lymphatic vessels
Article Snippet: .. In some experiments, explants were incubated in dispase II (Boehringer Mannheim) for 45 min at 37°C to separate the epidermis from dermis. ..

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

Mouse Assay:

Article Title: Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation
Article Snippet: .. Synovial tissue was dissected from knee joints of mice following induction of arthritis and digested in a cocktail of 2.4 mg/ml dispase (Boehringer Mannheim, Mannheim, Germany), 1 mg/ml collagenase (Sigma-Aldrich), and 100 μg/ml DNase I (Boehringer Mannheim) in RPMI for 1 hour at 37°C. .. The cells were filtered through a nylon mesh, washed three times in RPMI plus 10% FCS (Trace Bioscientific, Sydney, Australia), and stained for flow cytometric analysis.

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    Boehringer Mannheim dispase
    PECAM immunofluorescence of ES cell differentiation. <t>Dispase-treated</t> ES cells were plated onto tissue culture dishes on day 0. Representative plates were fixed every other day. Wells were stained with an anti-PECAM antibody and a B-phycoerythrin-conjugated secondary antibody. A, D : day 2; B, E : day 4; C, F : day 6; G, J : day 8; H, K : day 10; I, J : day 12. A–C, G–I : fluorescence photomicrograph; D–F, J–L : the corresponding phase contrast photomicrograph. Arrowheads in H point to PECAM strain at cell borders. Scale bar, 30 μm.
    Dispase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PECAM immunofluorescence of ES cell differentiation. Dispase-treated ES cells were plated onto tissue culture dishes on day 0. Representative plates were fixed every other day. Wells were stained with an anti-PECAM antibody and a B-phycoerythrin-conjugated secondary antibody. A, D : day 2; B, E : day 4; C, F : day 6; G, J : day 8; H, K : day 10; I, J : day 12. A–C, G–I : fluorescence photomicrograph; D–F, J–L : the corresponding phase contrast photomicrograph. Arrowheads in H point to PECAM strain at cell borders. Scale bar, 30 μm.

    Journal: The American Journal of Pathology

    Article Title: Developmental Platelet Endothelial Cell Adhesion Molecule Expression Suggests Multiple Roles for a Vascular Adhesion Molecule

    doi:

    Figure Lengend Snippet: PECAM immunofluorescence of ES cell differentiation. Dispase-treated ES cells were plated onto tissue culture dishes on day 0. Representative plates were fixed every other day. Wells were stained with an anti-PECAM antibody and a B-phycoerythrin-conjugated secondary antibody. A, D : day 2; B, E : day 4; C, F : day 6; G, J : day 8; H, K : day 10; I, J : day 12. A–C, G–I : fluorescence photomicrograph; D–F, J–L : the corresponding phase contrast photomicrograph. Arrowheads in H point to PECAM strain at cell borders. Scale bar, 30 μm.

    Article Snippet: These cultures were then dissociated into clumps by treatment with Dispase (Boehringer Mannheim, Indianapolis, IN) on day 0.

    Techniques: Immunofluorescence, Cell Differentiation, Staining, Fluorescence

    Double immunofluorescence for PECAM and CD34 in ES cells and during ES cell differentiation. A, F : undifferentiated ES cells, fixed 3 days after passage. B–E, G–J : Dispase-treated ES cells were cultured in Petri dishes for 3 days, then plated onto tissue culture dishes. Representative plates were fixed every day, beginning with day 5. Wells were stained with biotin-conjugated Mec 13.3 ( A–C, E ) and an anti-CD34 rat monoclonal antibody ( F–I ). Control plates had either the anti-PECAM primary antibody ( D ) or the anti-CD34 antibody primary antibody ( J ) omitted. Detection was with streptavidin-conjugated B-phycoerythrin and donkey anti-rat FITC. A, F , undifferentiated ES cells; B, G , day 6; C–E, H–J , day 10. A–E , PECAM-specific B-phycoerythrin fluorescence; F–J , CD34-specific FITC fluorescence of the same wells. In B and G , the arrow shows cells that are CD34-negative and PECAM-positive and the arrowhead indicates cells that are double positive.

    Journal: The American Journal of Pathology

    Article Title: Developmental Platelet Endothelial Cell Adhesion Molecule Expression Suggests Multiple Roles for a Vascular Adhesion Molecule

    doi:

    Figure Lengend Snippet: Double immunofluorescence for PECAM and CD34 in ES cells and during ES cell differentiation. A, F : undifferentiated ES cells, fixed 3 days after passage. B–E, G–J : Dispase-treated ES cells were cultured in Petri dishes for 3 days, then plated onto tissue culture dishes. Representative plates were fixed every day, beginning with day 5. Wells were stained with biotin-conjugated Mec 13.3 ( A–C, E ) and an anti-CD34 rat monoclonal antibody ( F–I ). Control plates had either the anti-PECAM primary antibody ( D ) or the anti-CD34 antibody primary antibody ( J ) omitted. Detection was with streptavidin-conjugated B-phycoerythrin and donkey anti-rat FITC. A, F , undifferentiated ES cells; B, G , day 6; C–E, H–J , day 10. A–E , PECAM-specific B-phycoerythrin fluorescence; F–J , CD34-specific FITC fluorescence of the same wells. In B and G , the arrow shows cells that are CD34-negative and PECAM-positive and the arrowhead indicates cells that are double positive.

    Article Snippet: These cultures were then dissociated into clumps by treatment with Dispase (Boehringer Mannheim, Indianapolis, IN) on day 0.

    Techniques: Immunofluorescence, Cell Differentiation, Cell Culture, Staining, Fluorescence

    Phenotype of cells accumulating in the synovium of SOCS-1 –/– IFN-γ –/– mice during acute arthritis. Synovium was dissected from the knee joints of SOCS-1 +/+ IFN-γ –/– and SOCS-1 –/– IFN-γ –/– mice on day 7 after arthritis induction, and synovial cells were isolated by digestion with dispase and collagenase. ( a ) Increased percentage of myeloid cells in the synovium of SOCS-1 –/– IFN-γ –/– mice during acute arthritis. Synovial cells were stained for expression of CD45, and analysis of CD11b and GR-1 expression was performed on CD45 + cells. Results shown are representative of three independent experiments. ( b ) Flow cytometric analysis of nonadherent synovial cells for expression of CD4 and CD8. Nonadherent synovial cells were isolated following overnight incubation. ( c ) Cytocentrifuge preparations of joint exudate cells, isolated on day 7 following arthritis induction. ×400.

    Journal: Journal of Clinical Investigation

    Article Title: Suppressor of cytokine signaling-1 regulates acute inflammatory arthritis and T cell activation

    doi: 10.1172/JCI200316156

    Figure Lengend Snippet: Phenotype of cells accumulating in the synovium of SOCS-1 –/– IFN-γ –/– mice during acute arthritis. Synovium was dissected from the knee joints of SOCS-1 +/+ IFN-γ –/– and SOCS-1 –/– IFN-γ –/– mice on day 7 after arthritis induction, and synovial cells were isolated by digestion with dispase and collagenase. ( a ) Increased percentage of myeloid cells in the synovium of SOCS-1 –/– IFN-γ –/– mice during acute arthritis. Synovial cells were stained for expression of CD45, and analysis of CD11b and GR-1 expression was performed on CD45 + cells. Results shown are representative of three independent experiments. ( b ) Flow cytometric analysis of nonadherent synovial cells for expression of CD4 and CD8. Nonadherent synovial cells were isolated following overnight incubation. ( c ) Cytocentrifuge preparations of joint exudate cells, isolated on day 7 following arthritis induction. ×400.

    Article Snippet: Synovial tissue was dissected from knee joints of mice following induction of arthritis and digested in a cocktail of 2.4 mg/ml dispase (Boehringer Mannheim, Mannheim, Germany), 1 mg/ml collagenase (Sigma-Aldrich), and 100 μg/ml DNase I (Boehringer Mannheim) in RPMI for 1 hour at 37°C.

    Techniques: Mouse Assay, Isolation, Staining, Expressing, Flow Cytometry, Incubation

    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

    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