dispase  (Thermo Fisher)


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    Name:
    Dispase II powder
    Description:
    Dispase II neutral protease is an amino endo peptidase that hydrolyzes the N terminal peptide bonds of non polar amino acid residues This sequence is found with high frequency in collagen Dispase II has a mild proteolytic action that makes it useful for the isolation and routine passaging of primary cells Dispase II is relatively gentle dissociates well at physiological temperature and pH and generally maintains cell membrane integrity Gibco Dispase II is produced in Bacillus polymyxa and packaged as a lyophilized non sterile powder for research use in cell or tissue dissociation and to prevent clumping in suspension cultures Gibco Dispase II activity is guaranteed to be greater than 0 5 units mg
    Catalog Number:
    17105041
    Price:
    None
    Applications:
    Cell Culture|Mammalian Cell Culture
    Category:
    Cell Culture Transfection Reagents
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    Structured Review

    Thermo Fisher dispase
    Post-detachment cell viability impacts the rate of cell expansion. (A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, <t>Dispase</t> or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10 5 viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10 5 and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.
    Dispase II neutral protease is an amino endo peptidase that hydrolyzes the N terminal peptide bonds of non polar amino acid residues This sequence is found with high frequency in collagen Dispase II has a mild proteolytic action that makes it useful for the isolation and routine passaging of primary cells Dispase II is relatively gentle dissociates well at physiological temperature and pH and generally maintains cell membrane integrity Gibco Dispase II is produced in Bacillus polymyxa and packaged as a lyophilized non sterile powder for research use in cell or tissue dissociation and to prevent clumping in suspension cultures Gibco Dispase II activity is guaranteed to be greater than 0 5 units mg
    https://www.bioz.com/result/dispase/product/Thermo Fisher
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    dispase - by Bioz Stars, 2021-01
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    Images

    1) Product Images from "Scalable Passaging of Adherent Human Pluripotent Stem Cells"

    Article Title: Scalable Passaging of Adherent Human Pluripotent Stem Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0088012

    Post-detachment cell viability impacts the rate of cell expansion. (A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, Dispase or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10 5 viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10 5 and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.
    Figure Legend Snippet: Post-detachment cell viability impacts the rate of cell expansion. (A) Comparison of cell numbers generated over 5 passages in mTeSR™1 on Matrigel™ using conventional colony scraping, Collagenase IV, Dispase or a 1 mM, 570 mOsmol/kg hypertonic sodium citrate solution to subculture the cells. Cell detachment methods were compared by continuously seeding 2×10 5 viable cells per well in six-well plates to control for differences in post-detachment cell recovery. When the hESC colonies for each condition reached confluence, cells were passaged and the viable number of cells determined. Three replicate wells were then individually re-plated at 2×10 5 and the process repeated. (B) The actual viable cell number determined at each passage was used to determine the total number of viable cells that would have been generated if all cells at each passage had been plated. Inset illustrates day 0 to day 15 with an expanded Y axis to illustrate the earlier passages. Error bars indicate standard error of the mean. All conditions, n = 3.

    Techniques Used: Generated

    2) Product Images from "Strain dependent differences in glucocorticoid-induced bone loss between C57BL/6J and CD-1 mice"

    Article Title: Strain dependent differences in glucocorticoid-induced bone loss between C57BL/6J and CD-1 mice

    Journal: Scientific Reports

    doi: 10.1038/srep36513

    In vitro osteogenesis of C57BL/6J and CD1 mouse calvarial cells in the presence of Dex. Calvarial OBL were isolated from 1 day old pups by collagenase II + Dispase enzymatic digestion and were plated in 96 well plates in osteogenic induction media with different Dex concentrations (0, 10^1, 10^2 and 10^3 nM). OBL mineralization was evaluated on day 6 by 1% alizarin red-S staining (Original magnification, x4, Pictures in upper row were obtained from CD1 mice and pictures in lower row were obtained from C57BL/6 mice) ( a ). The percentage of Alizarin red-positive bone nodules area reported to the total well area was quantified with Image-J software. Results are represented as mean of 4 (n = 4) independent experiments, each performed in triplicates ( b ). Error bars correspond to SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (*p
    Figure Legend Snippet: In vitro osteogenesis of C57BL/6J and CD1 mouse calvarial cells in the presence of Dex. Calvarial OBL were isolated from 1 day old pups by collagenase II + Dispase enzymatic digestion and were plated in 96 well plates in osteogenic induction media with different Dex concentrations (0, 10^1, 10^2 and 10^3 nM). OBL mineralization was evaluated on day 6 by 1% alizarin red-S staining (Original magnification, x4, Pictures in upper row were obtained from CD1 mice and pictures in lower row were obtained from C57BL/6 mice) ( a ). The percentage of Alizarin red-positive bone nodules area reported to the total well area was quantified with Image-J software. Results are represented as mean of 4 (n = 4) independent experiments, each performed in triplicates ( b ). Error bars correspond to SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparison test (*p

    Techniques Used: In Vitro, Isolation, Staining, Mouse Assay, Software

    3) Product Images from "A New Technique for Primary Hepatocyte Expansion In Vitro"

    Article Title: A New Technique for Primary Hepatocyte Expansion In Vitro

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.21911

    Morphology and function of hepatocytes in subcultures. Hepatocytes were cocultured with 3T3-J2 fibroblasts for 35 days, detached from the substrate by dispase/collagenase treatment, dispersed in a single cell suspension by trypsin digestion, replated,
    Figure Legend Snippet: Morphology and function of hepatocytes in subcultures. Hepatocytes were cocultured with 3T3-J2 fibroblasts for 35 days, detached from the substrate by dispase/collagenase treatment, dispersed in a single cell suspension by trypsin digestion, replated,

    Techniques Used:

    4) Product Images from "Cadherin Cytoplasmic Domains Inhibit the Cell Surface Localization of Endogenous E-Cadherin, Blocking Desmosome and Tight Junction Formation and Inducing Cell Dissociation"

    Article Title: Cadherin Cytoplasmic Domains Inhibit the Cell Surface Localization of Endogenous E-Cadherin, Blocking Desmosome and Tight Junction Formation and Inducing Cell Dissociation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0105313

    Expression of the DsRed-tagged E-cadherin cytoplasmic domain in MDCK cells disrupts cell–cell adhesion. (A) Schematic representation of DsRed-tagged cadherin cytoplasmic domain constructs. DECT is a DsRed-tagged wild-type E-cadherin cytoplasmic domain (ECT). The binding sites for p120 and β-catenin/plakoglobin (β-cat/plako) are shown. DECTEA is an ECT construct with alanine substitutions of the two conserved glutamic acid residues and a conserved aspartic acid residue (Glu-Glu-Asp) in the p120-binding site, which has been shown to eliminate the interaction with p120. DECTSA is an ECT construct with alanine substitutions of the conserved eight serine residues in the catenin-binding site, which has been shown to weaken the interaction with β-catenin. DECTN is a chimeric construct composed of DsRed and the N-terminal region of ECT containing the p120-catenin–binding site. DECTC is a chimera of DsRed and the C-terminal half of ECT containing the catenin–binding site. DNCT is an N-cadherin cytoplasmic domain (NCT) construct. The C-terminus of all constructs, including DsRed, is tagged with the FLAG epitope. (B) Morphology of MDCK cells expressing DsRed, DECT, and Snail. DECT+ and Snail+ cells lose cell–cell contacts. (C) The migration assay. While MDCK cells expressing Snail or DECT show enhanced migration, MDCK cells expressing DsRed do not. The results are represented as the mean ± SD of three independent experiments. (D) Dissociation assays. Cells were incubated with dispase and detached cells were subjected to mechanical stress by pipetting as described in Materials and Methods. (E) Immunoblot analysis revealed that up-regulation of fibronectin, N-cadherin, and vimentin and down-regulation of E-cadherin and occludin occurred in Snail+ cells but not in DECT+ cells. Vinculin was used as a loading control. (F) Invasion assays. Representative photographs of the cells that invaded (Snail+) and not invaded (parental MDCK) Matrigel (upper panels). The results are represented as the mean ± SD of three independent experiments. Bars, 25 µm.
    Figure Legend Snippet: Expression of the DsRed-tagged E-cadherin cytoplasmic domain in MDCK cells disrupts cell–cell adhesion. (A) Schematic representation of DsRed-tagged cadherin cytoplasmic domain constructs. DECT is a DsRed-tagged wild-type E-cadherin cytoplasmic domain (ECT). The binding sites for p120 and β-catenin/plakoglobin (β-cat/plako) are shown. DECTEA is an ECT construct with alanine substitutions of the two conserved glutamic acid residues and a conserved aspartic acid residue (Glu-Glu-Asp) in the p120-binding site, which has been shown to eliminate the interaction with p120. DECTSA is an ECT construct with alanine substitutions of the conserved eight serine residues in the catenin-binding site, which has been shown to weaken the interaction with β-catenin. DECTN is a chimeric construct composed of DsRed and the N-terminal region of ECT containing the p120-catenin–binding site. DECTC is a chimera of DsRed and the C-terminal half of ECT containing the catenin–binding site. DNCT is an N-cadherin cytoplasmic domain (NCT) construct. The C-terminus of all constructs, including DsRed, is tagged with the FLAG epitope. (B) Morphology of MDCK cells expressing DsRed, DECT, and Snail. DECT+ and Snail+ cells lose cell–cell contacts. (C) The migration assay. While MDCK cells expressing Snail or DECT show enhanced migration, MDCK cells expressing DsRed do not. The results are represented as the mean ± SD of three independent experiments. (D) Dissociation assays. Cells were incubated with dispase and detached cells were subjected to mechanical stress by pipetting as described in Materials and Methods. (E) Immunoblot analysis revealed that up-regulation of fibronectin, N-cadherin, and vimentin and down-regulation of E-cadherin and occludin occurred in Snail+ cells but not in DECT+ cells. Vinculin was used as a loading control. (F) Invasion assays. Representative photographs of the cells that invaded (Snail+) and not invaded (parental MDCK) Matrigel (upper panels). The results are represented as the mean ± SD of three independent experiments. Bars, 25 µm.

    Techniques Used: Expressing, Construct, Binding Assay, FLAG-tag, Migration, Incubation

    5) Product Images from "Genomic programming of IRF4-expressing human Langerhans cells"

    Article Title: Genomic programming of IRF4-expressing human Langerhans cells

    Journal: Nature Communications

    doi: 10.1038/s41467-019-14125-x

    System for analysing human LCs and control of antigen cross-presentation. a Schematic depicting isolation of primary human LCs. Split healthy skin was treated with dispase for 20 h to dissociate epidermis. Steady-state LCs were isolated from the epidermis by digestion with liberase TM or migrated from the epidermal sheets for 48 h in cell culture medium and stimulated with TNF (24 h) to induce their activation. b Flow cytometry assessment of steady-state and migrated LC. LCs were enumerated as CD207/CD1a/HLA-DR high cells. A representative example of n > 5 independent donors. c Flow cytometry assessment of activation markers expressed by steady-state and migrated LC. Co-stimulatory molecules critical for T-cell activation during antigen presentation (CD70, CD86 and CD40) were analysed in CD207/CD1a/HLA-DR high cells. A representative example of n > 5 independent donors. d IFN-γ secretion by an EBV-specific CD8 T-cell line stimulated with antigen presenting LCs in the context of MHC-I HLA-A2. Steady-state or migrated LCs were pulsed with 30-amino acid peptides containing EBV epitope (dark grey). Pulsed or unpulsed (light grey) LCs were stimulated with TNF and then assayed for IFN-γ secretion. ELISpot assay, n = 5 independent experiments in triplicate, paired t test, box and whiskers show min and max value, line at median. Source data are provided as a Source Data file. e IFN-γ secretion by EBV-specific CD8 T-cell line stimulated by migrated LCs pulsed as in panel d . IFN-γ secretion was measured with (black) or without (grey) TNF stimulation. ELISpot assay, n = 5 independent experiments in triplicate, paired t test, box and whiskers show min and max value, line at median. Source data are provided as a Source Data file.
    Figure Legend Snippet: System for analysing human LCs and control of antigen cross-presentation. a Schematic depicting isolation of primary human LCs. Split healthy skin was treated with dispase for 20 h to dissociate epidermis. Steady-state LCs were isolated from the epidermis by digestion with liberase TM or migrated from the epidermal sheets for 48 h in cell culture medium and stimulated with TNF (24 h) to induce their activation. b Flow cytometry assessment of steady-state and migrated LC. LCs were enumerated as CD207/CD1a/HLA-DR high cells. A representative example of n > 5 independent donors. c Flow cytometry assessment of activation markers expressed by steady-state and migrated LC. Co-stimulatory molecules critical for T-cell activation during antigen presentation (CD70, CD86 and CD40) were analysed in CD207/CD1a/HLA-DR high cells. A representative example of n > 5 independent donors. d IFN-γ secretion by an EBV-specific CD8 T-cell line stimulated with antigen presenting LCs in the context of MHC-I HLA-A2. Steady-state or migrated LCs were pulsed with 30-amino acid peptides containing EBV epitope (dark grey). Pulsed or unpulsed (light grey) LCs were stimulated with TNF and then assayed for IFN-γ secretion. ELISpot assay, n = 5 independent experiments in triplicate, paired t test, box and whiskers show min and max value, line at median. Source data are provided as a Source Data file. e IFN-γ secretion by EBV-specific CD8 T-cell line stimulated by migrated LCs pulsed as in panel d . IFN-γ secretion was measured with (black) or without (grey) TNF stimulation. ELISpot assay, n = 5 independent experiments in triplicate, paired t test, box and whiskers show min and max value, line at median. Source data are provided as a Source Data file.

    Techniques Used: Isolation, Cell Culture, Activation Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunospot

    6) Product Images from "Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin"

    Article Title: Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin

    Journal: Biology Open

    doi: 10.1242/bio.031369

    The α-catenin construct α1–381 requires vinculin for its ability to rescue junction formation. Using the CRISPR/Cas9 system, the vinculin gene was ablated in α1–381/GFP–IIA/IIAKO cells, yielding α1–381/GFP–IIA/IIAKO-vincKO double-knockout cells. (A) Immunoblot detection of vinculin (vinc) and actin. α1–381/GFP–IIA/IIAKO cells with wild-type vinculin (vincWT) were used as a positive control, and actin was used as a loading control. (B) Genomic sequencing revealed a mutation in the target region of vinculin gene that induced a frame-shift. All 13 independent clones sequenced harbored the same insertion of 1 bp. (C) Morphology of α1–381/GFP–IIA/IIAKO-vincKO cells. Cells cultured in the presence of Dox were observed using a phase-contrast microscope. (D) Cells cultured in the presence of Dox were incubated with Dispase, and then detached cells were subjected to dissociation assays and quantified. Values represent the mean±s.e.; n =at least 3 times. * P
    Figure Legend Snippet: The α-catenin construct α1–381 requires vinculin for its ability to rescue junction formation. Using the CRISPR/Cas9 system, the vinculin gene was ablated in α1–381/GFP–IIA/IIAKO cells, yielding α1–381/GFP–IIA/IIAKO-vincKO double-knockout cells. (A) Immunoblot detection of vinculin (vinc) and actin. α1–381/GFP–IIA/IIAKO cells with wild-type vinculin (vincWT) were used as a positive control, and actin was used as a loading control. (B) Genomic sequencing revealed a mutation in the target region of vinculin gene that induced a frame-shift. All 13 independent clones sequenced harbored the same insertion of 1 bp. (C) Morphology of α1–381/GFP–IIA/IIAKO-vincKO cells. Cells cultured in the presence of Dox were observed using a phase-contrast microscope. (D) Cells cultured in the presence of Dox were incubated with Dispase, and then detached cells were subjected to dissociation assays and quantified. Values represent the mean±s.e.; n =at least 3 times. * P

    Techniques Used: Construct, CRISPR, Double Knockout, Positive Control, Genomic Sequencing, Mutagenesis, Clone Assay, Cell Culture, Microscopy, Incubation

    Expression of α-catenin deletion constructs in IIAKO cells induces junction assembly and restores cell–cell adhesion. (A) Schematic representation of α-catenin (top) and mutant derivatives (bottom). The N-terminal (N) domain binds to β-catenin. The middle (M) domain comprises three regions, MI, MII, and MIII. MI contains binding sites for vinculin, and MII and MIII are involved in regulation of force-dependent vinculin binding. The C-terminal (C) domain binds actin. α1–302 does not contain the vinculin- and actin-binding sites. α1–381 is a construct in which the carboxy-terminal 525 residues have been truncated, thereby removing the actin-binding site but retaining the vinculin-binding site. αΔ203–612 has an internal deletion of residues 203–612, and therefore lacks the vinculin-binding site but retains the actin-binding site. All constructs were tagged with HA. (B) Immunoblot detection of α-catenin polypeptides. GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612 were cultured in the presence or absence of Dox for 4 days, and then analyzed. The blots were probed with anti-HA, anti-GFP, and anti-vinculin antibodies. (C) Immunofluorescence staining with anti-HA revealed that these constructs were localized at cell–cell contact sites of cells cultured in the absence of Dox. (D) Morphology of GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612. Cells cultured in the presence of Dox to suppress GFP–IIA expression, and were observed using a phase contrast microscope. (E) Dissociation assays. Cells were incubated with Dispase, and then detached cells were subjected to mechanical stress by pipetting and quantified. Values represent the mean±s.e.; n =at least 3 times. * P
    Figure Legend Snippet: Expression of α-catenin deletion constructs in IIAKO cells induces junction assembly and restores cell–cell adhesion. (A) Schematic representation of α-catenin (top) and mutant derivatives (bottom). The N-terminal (N) domain binds to β-catenin. The middle (M) domain comprises three regions, MI, MII, and MIII. MI contains binding sites for vinculin, and MII and MIII are involved in regulation of force-dependent vinculin binding. The C-terminal (C) domain binds actin. α1–302 does not contain the vinculin- and actin-binding sites. α1–381 is a construct in which the carboxy-terminal 525 residues have been truncated, thereby removing the actin-binding site but retaining the vinculin-binding site. αΔ203–612 has an internal deletion of residues 203–612, and therefore lacks the vinculin-binding site but retains the actin-binding site. All constructs were tagged with HA. (B) Immunoblot detection of α-catenin polypeptides. GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612 were cultured in the presence or absence of Dox for 4 days, and then analyzed. The blots were probed with anti-HA, anti-GFP, and anti-vinculin antibodies. (C) Immunofluorescence staining with anti-HA revealed that these constructs were localized at cell–cell contact sites of cells cultured in the absence of Dox. (D) Morphology of GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612. Cells cultured in the presence of Dox to suppress GFP–IIA expression, and were observed using a phase contrast microscope. (E) Dissociation assays. Cells were incubated with Dispase, and then detached cells were subjected to mechanical stress by pipetting and quantified. Values represent the mean±s.e.; n =at least 3 times. * P

    Techniques Used: Expressing, Construct, Mutagenesis, Binding Assay, Cell Culture, Immunofluorescence, Staining, Microscopy, Incubation

    7) Product Images from "Ocular pathogenesis and immune reaction after intravitreal dispase injection in mice"

    Article Title: Ocular pathogenesis and immune reaction after intravitreal dispase injection in mice

    Journal: Molecular Vision

    doi:

    Immunofluorescence analysis showed no cluster of differentiation (CD)3+ labeled T cells, F4/80+ labeled macrophages, or CD56+ labeled natural killer (NK) cells involved in dispase-injected eyes at 48 h time point ( A , B ). Bluish cells stained with Hoechst 33342 (scale bar 100 μm).
    Figure Legend Snippet: Immunofluorescence analysis showed no cluster of differentiation (CD)3+ labeled T cells, F4/80+ labeled macrophages, or CD56+ labeled natural killer (NK) cells involved in dispase-injected eyes at 48 h time point ( A , B ). Bluish cells stained with Hoechst 33342 (scale bar 100 μm).

    Techniques Used: Immunofluorescence, Labeling, Injection, Staining

    Intracellular staining at the 4-week time point in single T cell suspensions from dispase-injected PVR-model mouse spleens. A : IFN-γ+/CD4+T cells 0.061% of the total CD4+T cells. B : IFN-γ+/CD8+T cells 0.23% of the total CD8+T cells.
    Figure Legend Snippet: Intracellular staining at the 4-week time point in single T cell suspensions from dispase-injected PVR-model mouse spleens. A : IFN-γ+/CD4+T cells 0.061% of the total CD4+T cells. B : IFN-γ+/CD8+T cells 0.23% of the total CD8+T cells.

    Techniques Used: Staining, Injection

    Dispase-injected mice developed cardinal features of proliferative vitreoretinopathy (PVR). A : Ocular fundi of dispase-injected eye and saline-injected control eye. Arrow shows PVR membranes. B : Hematoxylin and eosin (H E) staining of dispase-injected PVR eye and saline-injected control eye. Arrow shows proliferative epiretinal membranes (EM). C : PVR percentages at 1, 2, 4, 6, and 8-week time points after dispase injection.
    Figure Legend Snippet: Dispase-injected mice developed cardinal features of proliferative vitreoretinopathy (PVR). A : Ocular fundi of dispase-injected eye and saline-injected control eye. Arrow shows PVR membranes. B : Hematoxylin and eosin (H E) staining of dispase-injected PVR eye and saline-injected control eye. Arrow shows proliferative epiretinal membranes (EM). C : PVR percentages at 1, 2, 4, 6, and 8-week time points after dispase injection.

    Techniques Used: Injection, Mouse Assay, Staining

    Electroretinograms (ERG) of the dispase-injected proliferative vitreoretinopathy (PVR) model eyes and saline-injected control eyes. Scotopic ERGs showed significant decreases in b-wave amplitudes, but no decreases in a-wave amplitudes of dispase-injected PVR eyes compared with saline-injected control eyes.
    Figure Legend Snippet: Electroretinograms (ERG) of the dispase-injected proliferative vitreoretinopathy (PVR) model eyes and saline-injected control eyes. Scotopic ERGs showed significant decreases in b-wave amplitudes, but no decreases in a-wave amplitudes of dispase-injected PVR eyes compared with saline-injected control eyes.

    Techniques Used: Injection

    Early inflammatory infiltration profile (0–5 day time points) in diapase-injected and saline-injected control mice. Panels A - G shows inflammatory infiltration profile 0–5 days. Panel H shows a control. Neutrophils appeared from hour 8 ( C ) to hour 48 ( F ) in the anterior chamber of dispase-injected eyes but not saline-injected eyes (hematoxylin and eosin [H E] staining, scale bar 100 μm). All the arrows of panels C - F shows neutrophils.
    Figure Legend Snippet: Early inflammatory infiltration profile (0–5 day time points) in diapase-injected and saline-injected control mice. Panels A - G shows inflammatory infiltration profile 0–5 days. Panel H shows a control. Neutrophils appeared from hour 8 ( C ) to hour 48 ( F ) in the anterior chamber of dispase-injected eyes but not saline-injected eyes (hematoxylin and eosin [H E] staining, scale bar 100 μm). All the arrows of panels C - F shows neutrophils.

    Techniques Used: Injection, Mouse Assay, Staining

    Enzyme-Linked ImmunoSpot (ELISpot) assay of interferon (INF) -γ in single T cell suspensions from dispase-injected and saline-injected mouse spleens. A : Compared with positive control group (stimulated with phytohemagglutinin, PHA), IFN-γ values were only detected at 4-week and 8-week time points in dispase-injected mice, and not detected at any time point in saline-injected mice. B : In the graph of counting statistics, there were only 25.5 points at the 4-week time point, and 2.5 points in the 8-week for dispase-injected mice.
    Figure Legend Snippet: Enzyme-Linked ImmunoSpot (ELISpot) assay of interferon (INF) -γ in single T cell suspensions from dispase-injected and saline-injected mouse spleens. A : Compared with positive control group (stimulated with phytohemagglutinin, PHA), IFN-γ values were only detected at 4-week and 8-week time points in dispase-injected mice, and not detected at any time point in saline-injected mice. B : In the graph of counting statistics, there were only 25.5 points at the 4-week time point, and 2.5 points in the 8-week for dispase-injected mice.

    Techniques Used: Enzyme-linked Immunospot, Injection, Positive Control, Mouse Assay

    8) Product Images from "Mtss1 Promotes Cell-Cell Junction Assembly and Stability through the Small GTPase Rac1"

    Article Title: Mtss1 Promotes Cell-Cell Junction Assembly and Stability through the Small GTPase Rac1

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0031141

    Mtss1 regulates cell-cell junction strength and inhibits HGF-scattering. (A) Mtss1 construct expression in Scc9 cells. Mtss1-GFP is approximately 3-fold over-expressed vs endogenous Mtss1 (estimated based on densitometry average from 3 experiments). (B) Small colonies of cells were incubated with 10 ng/ml HGF for 20 hours and still photos are shown from live timelapse ( Movie S1 ). Graph is % colonies scattering n = 3 experiments. (C) Immunofluorescence labeling of E-cadherin and F-actin following HGF-induced cell scatter for 0, 3 and 6 hours in SCC9 control cells, Mtss1 expressing or K4D mutant expressing as indicated. Images representative of each time point. (D) Effect of HGF treatment on the average number of junctions labeled with E-cadherin per colony is shown relative to Scc9 cells. 40+ colonies were counted per cell line, per experiment (n = 3). (E) Number of single cells released in dispase assay assay (n = 6 experiments). For D E Mean ± S.E.M. *** p
    Figure Legend Snippet: Mtss1 regulates cell-cell junction strength and inhibits HGF-scattering. (A) Mtss1 construct expression in Scc9 cells. Mtss1-GFP is approximately 3-fold over-expressed vs endogenous Mtss1 (estimated based on densitometry average from 3 experiments). (B) Small colonies of cells were incubated with 10 ng/ml HGF for 20 hours and still photos are shown from live timelapse ( Movie S1 ). Graph is % colonies scattering n = 3 experiments. (C) Immunofluorescence labeling of E-cadherin and F-actin following HGF-induced cell scatter for 0, 3 and 6 hours in SCC9 control cells, Mtss1 expressing or K4D mutant expressing as indicated. Images representative of each time point. (D) Effect of HGF treatment on the average number of junctions labeled with E-cadherin per colony is shown relative to Scc9 cells. 40+ colonies were counted per cell line, per experiment (n = 3). (E) Number of single cells released in dispase assay assay (n = 6 experiments). For D E Mean ± S.E.M. *** p

    Techniques Used: Construct, Expressing, Incubation, Immunofluorescence, Labeling, Mutagenesis

    9) Product Images from "Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant"

    Article Title: Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06151-y

    RF induces low-level local inflammation. Dorsal skin of C57BL/6 mice were exposed to RF or intradermally injected with 20 µl Alum (1:1 volume ratio in PBS), AddaVax (50%, vol/vol in PBS), or MPL (25 µg). Adjuvant-treated and non-treated skins were dissected at indicated times. a , b Heat map of relative cytokine ( a ) and chemokine ( b ) gene expression. Total RNA was extracted followed by reverse transcription and real-time PCR analysis of cytokine and chemokine gene expression using GAPDH as an internal control. The baseline gene expression level was set at 1. c – g Different innate immune cell levels in RF and adjuvant-treated skin. Skin was digested in collagenase D and dispase to prepare single-cell suspensions. Cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis of levels of different cell types: neutrophils ( c ), monocytes ( d ), macrophages ( e ), eosinophils ( f ), and mDCs ( g ) (Supplementary Fig. 2). n = 4. Student’s t -test was used to compare differences between groups at 48 and 96 h. *, p
    Figure Legend Snippet: RF induces low-level local inflammation. Dorsal skin of C57BL/6 mice were exposed to RF or intradermally injected with 20 µl Alum (1:1 volume ratio in PBS), AddaVax (50%, vol/vol in PBS), or MPL (25 µg). Adjuvant-treated and non-treated skins were dissected at indicated times. a , b Heat map of relative cytokine ( a ) and chemokine ( b ) gene expression. Total RNA was extracted followed by reverse transcription and real-time PCR analysis of cytokine and chemokine gene expression using GAPDH as an internal control. The baseline gene expression level was set at 1. c – g Different innate immune cell levels in RF and adjuvant-treated skin. Skin was digested in collagenase D and dispase to prepare single-cell suspensions. Cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis of levels of different cell types: neutrophils ( c ), monocytes ( d ), macrophages ( e ), eosinophils ( f ), and mDCs ( g ) (Supplementary Fig. 2). n = 4. Student’s t -test was used to compare differences between groups at 48 and 96 h. *, p

    Techniques Used: Mouse Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Flow Cytometry, Cytometry

    RF increases antigen uptake of DCs in skin. Lateral back skin of C57BL/6 mice was exposed to RF or sham treatment followed by ID injection of 2 µg AF647-OVA into RF or sham-treated skin. ID injection of PBS served as control. Skin was harvested 24 h later and digested in collagenase D and dispase to prepare single-cell suspensions. Skin cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis. a Live cells were gated and plotted based on CD11c and MHC II expression. CD11c + MHC II + cells were gated and plotted based on Langerin expression. b Langerin + cells (gate 1) were plotted based on CD11b and CD103 expression into Langerin + CD11b − CD103 + (I) and Langerin + CD11b + CD103 − subsets (II), whereas Langerin − cells (gate 2) were plotted based on CD11b expression into Langerin − CD11b + (III) and Langerin − CD11b − subsets (IV). c Percentage of AF647 + cells was analyzed for each DC subset. n = 4 for PBS control and 6 for sham and RF groups. Student’s t -test was used to compare differences between RF and sham groups. * p
    Figure Legend Snippet: RF increases antigen uptake of DCs in skin. Lateral back skin of C57BL/6 mice was exposed to RF or sham treatment followed by ID injection of 2 µg AF647-OVA into RF or sham-treated skin. ID injection of PBS served as control. Skin was harvested 24 h later and digested in collagenase D and dispase to prepare single-cell suspensions. Skin cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis. a Live cells were gated and plotted based on CD11c and MHC II expression. CD11c + MHC II + cells were gated and plotted based on Langerin expression. b Langerin + cells (gate 1) were plotted based on CD11b and CD103 expression into Langerin + CD11b − CD103 + (I) and Langerin + CD11b + CD103 − subsets (II), whereas Langerin − cells (gate 2) were plotted based on CD11b expression into Langerin − CD11b + (III) and Langerin − CD11b − subsets (IV). c Percentage of AF647 + cells was analyzed for each DC subset. n = 4 for PBS control and 6 for sham and RF groups. Student’s t -test was used to compare differences between RF and sham groups. * p

    Techniques Used: Mouse Assay, Injection, Staining, Fluorescence, Flow Cytometry, Cytometry, Expressing

    10) Product Images from "Diverse hematopoietic potentials of five human embryonic stem cell lines"

    Article Title: Diverse hematopoietic potentials of five human embryonic stem cell lines

    Journal: Experimental cell research

    doi: 10.1016/j.yexcr.2008.07.019

    Undifferentiated phenotype of five human embryonic stem cell (hESC) lines. hESC lines on feeders were treated with dispase until the colonies were completely detached from the feeder layers. Colonies were collected, washed, and made into single cells by trypsin digestion. Cells were stained with SSEA-3 or SSEA-4, followed by corresponding secondary antibodies. Filled lines represent samples stained with both primary and secondary antibodies. Open lines represent samples stained with secondary antibodies only. M1 shows the gating for positive cells whereas the number above the gate represents the percentage of positive cells. The staining of alkaline phosphatase activity and Oct-4 expression was performed on a 24-well plate as stated in the Materials and methods. Images were obtained using a Leica DMIL inverted microscope (Leica, Heidelberg, Germany) with a Leica C PLAN 4×/0.10 numeric aperture objective and a PixeLINK megapixel FireWire camera (model PL-A662; PixeLINK, Ottawa, ON, Canada) with PixeLINK Capture software version 1.0.
    Figure Legend Snippet: Undifferentiated phenotype of five human embryonic stem cell (hESC) lines. hESC lines on feeders were treated with dispase until the colonies were completely detached from the feeder layers. Colonies were collected, washed, and made into single cells by trypsin digestion. Cells were stained with SSEA-3 or SSEA-4, followed by corresponding secondary antibodies. Filled lines represent samples stained with both primary and secondary antibodies. Open lines represent samples stained with secondary antibodies only. M1 shows the gating for positive cells whereas the number above the gate represents the percentage of positive cells. The staining of alkaline phosphatase activity and Oct-4 expression was performed on a 24-well plate as stated in the Materials and methods. Images were obtained using a Leica DMIL inverted microscope (Leica, Heidelberg, Germany) with a Leica C PLAN 4×/0.10 numeric aperture objective and a PixeLINK megapixel FireWire camera (model PL-A662; PixeLINK, Ottawa, ON, Canada) with PixeLINK Capture software version 1.0.

    Techniques Used: Staining, Activity Assay, Expressing, Inverted Microscopy, Software

    11) Product Images from "Retinal Organoids derived from hiPSCs of an AIPL1-LCA Patient Maintain Cytoarchitecture despite Reduced levels of Mutant AIPL1"

    Article Title: Retinal Organoids derived from hiPSCs of an AIPL1-LCA Patient Maintain Cytoarchitecture despite Reduced levels of Mutant AIPL1

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-62047-2

    Generation of 3D AIPL1-LCA ROs from Patient hiPSCs. ( A ) Schematic of the differentiation protocol. ( B ) Phase contrast micrographs of differentiation stages: hiPSCs, floating aggregates of hiPSCs following treatment with dispase (W2), and aggregates plated on growth factor reduced (GFR) Matrigel-coated plates reach a typical morphology by week 4 (W4) are dissected manually and are grown in suspension after that (W4’). The typical transparent neuroepithelial domain (*) is formed (W12) with stratified appearance. Inset at larger magnification is shown (W12'). At W20 the projections at the surface begin to emerge (inset, arrowhead). Scale bars: 200 µm. ( C ) By week 23 (W23) the ROs reached 1–1.5 mm in diameter and displayed dense translucent projections at the apical edge (black arrowhead) that grow after that (W26). The double arrow shows the presumptive ONL. Abbreviations: 3D, three-dimensional; RO, retinal organoids; GFR, growth factor reduced; W, week; Tau, taurine; RA, retinoic acid; FBS, fetal bovine serum; ULA, ultra-low attachment plates; ONL, outer nuclear layer.
    Figure Legend Snippet: Generation of 3D AIPL1-LCA ROs from Patient hiPSCs. ( A ) Schematic of the differentiation protocol. ( B ) Phase contrast micrographs of differentiation stages: hiPSCs, floating aggregates of hiPSCs following treatment with dispase (W2), and aggregates plated on growth factor reduced (GFR) Matrigel-coated plates reach a typical morphology by week 4 (W4) are dissected manually and are grown in suspension after that (W4’). The typical transparent neuroepithelial domain (*) is formed (W12) with stratified appearance. Inset at larger magnification is shown (W12'). At W20 the projections at the surface begin to emerge (inset, arrowhead). Scale bars: 200 µm. ( C ) By week 23 (W23) the ROs reached 1–1.5 mm in diameter and displayed dense translucent projections at the apical edge (black arrowhead) that grow after that (W26). The double arrow shows the presumptive ONL. Abbreviations: 3D, three-dimensional; RO, retinal organoids; GFR, growth factor reduced; W, week; Tau, taurine; RA, retinoic acid; FBS, fetal bovine serum; ULA, ultra-low attachment plates; ONL, outer nuclear layer.

    Techniques Used:

    12) Product Images from "Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture"

    Article Title: Presence of native limbal stromal cells increases the expansion efficiency of limbal stem/progenitor cells in culture

    Journal: Experimental eye research

    doi: 10.1016/j.exer.2013.08.020

    3.2. Comparison of the limbal epithelial cells isolated by dispase II and ColA digestion
    Figure Legend Snippet: 3.2. Comparison of the limbal epithelial cells isolated by dispase II and ColA digestion

    Techniques Used: Isolation

    13) Product Images from "GPR39 marks specific cells within the sebaceous gland and contributes to skin wound healing"

    Article Title: GPR39 marks specific cells within the sebaceous gland and contributes to skin wound healing

    Journal: Scientific Reports

    doi: 10.1038/srep07913

    Localisation of GPR39 protein expression in HFs. ( a ) Immunostaining of GPR39 (green) in the HF and SG. PI was used for counterstaining (red). ( b ) Costaining of GPR39 (red) and Blimp1 (green) in the HF. Hoechst was used for counterstaining (blue). Inserted in the panel are separate colour channels and an enlarged picture of the SG section. ( c ) Costaining of GPR39 (red) and Lrig1 (green) in the HF. ( d ) SGs detached from the tail skin after dispase digestion. ( e ) Immunostaining of GPR39 in detached SGs. PI was used for counter staining (red). ( f ) Costaining of GPR39 (red) and Blimp1 (green) in a detached SG. The cells were counterstained with Hoechst (blue). ( g ) Flow cytometric analysis of epidermal cells using a PE-conjugated GPR39 antibody. The upper panel shows cells stained with the GPR39 antibody, and the lower panel shows the negative control without antibody staining. Inserted in the upper panel is an image of a sorted cell with PE fluorescence. ( h ) Expression of GPR39 in a human hair follicle. Arrows indicate positive signals. HF, hair follicle; SG, sebaceous gland; Epi, epidermis. Bar ( a ) = 25 μm, ( b, c ) = 20 μm, ( d ) = 5 μm, ( e, f, g, h ) = 20 μm.
    Figure Legend Snippet: Localisation of GPR39 protein expression in HFs. ( a ) Immunostaining of GPR39 (green) in the HF and SG. PI was used for counterstaining (red). ( b ) Costaining of GPR39 (red) and Blimp1 (green) in the HF. Hoechst was used for counterstaining (blue). Inserted in the panel are separate colour channels and an enlarged picture of the SG section. ( c ) Costaining of GPR39 (red) and Lrig1 (green) in the HF. ( d ) SGs detached from the tail skin after dispase digestion. ( e ) Immunostaining of GPR39 in detached SGs. PI was used for counter staining (red). ( f ) Costaining of GPR39 (red) and Blimp1 (green) in a detached SG. The cells were counterstained with Hoechst (blue). ( g ) Flow cytometric analysis of epidermal cells using a PE-conjugated GPR39 antibody. The upper panel shows cells stained with the GPR39 antibody, and the lower panel shows the negative control without antibody staining. Inserted in the upper panel is an image of a sorted cell with PE fluorescence. ( h ) Expression of GPR39 in a human hair follicle. Arrows indicate positive signals. HF, hair follicle; SG, sebaceous gland; Epi, epidermis. Bar ( a ) = 25 μm, ( b, c ) = 20 μm, ( d ) = 5 μm, ( e, f, g, h ) = 20 μm.

    Techniques Used: Expressing, Immunostaining, Staining, Flow Cytometry, Negative Control, Fluorescence

    14) Product Images from "Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation"

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation

    Journal: PeerJ

    doi: 10.7717/peerj.224

    Embryoid body derivation from an mRFP1-negative iPSC clone. IPSCs growing in log phase were released with Dispase treatment and allowed to form embryoid bodies in low-attachment flasks as described in Materials and Methods. The flasks were observed periodically (at the indicated intervals) under the microscope and photographed.
    Figure Legend Snippet: Embryoid body derivation from an mRFP1-negative iPSC clone. IPSCs growing in log phase were released with Dispase treatment and allowed to form embryoid bodies in low-attachment flasks as described in Materials and Methods. The flasks were observed periodically (at the indicated intervals) under the microscope and photographed.

    Techniques Used: Microscopy

    15) Product Images from "Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant"

    Article Title: Augmentation of vaccine-induced humoral and cellular immunity by a physical radiofrequency adjuvant

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06151-y

    RF induces low-level local inflammation. Dorsal skin of C57BL/6 mice were exposed to RF or intradermally injected with 20 µl Alum (1:1 volume ratio in PBS), AddaVax (50%, vol/vol in PBS), or MPL (25 µg). Adjuvant-treated and non-treated skins were dissected at indicated times. a , b Heat map of relative cytokine ( a ) and chemokine ( b ) gene expression. Total RNA was extracted followed by reverse transcription and real-time PCR analysis of cytokine and chemokine gene expression using GAPDH as an internal control. The baseline gene expression level was set at 1. c – g Different innate immune cell levels in RF and adjuvant-treated skin. Skin was digested in collagenase D and dispase to prepare single-cell suspensions. Cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis of levels of different cell types: neutrophils ( c ), monocytes ( d ), macrophages ( e ), eosinophils ( f ), and mDCs ( g ) (Supplementary Fig. 2). n = 4. Student’s t -test was used to compare differences between groups at 48 and 96 h. *, p
    Figure Legend Snippet: RF induces low-level local inflammation. Dorsal skin of C57BL/6 mice were exposed to RF or intradermally injected with 20 µl Alum (1:1 volume ratio in PBS), AddaVax (50%, vol/vol in PBS), or MPL (25 µg). Adjuvant-treated and non-treated skins were dissected at indicated times. a , b Heat map of relative cytokine ( a ) and chemokine ( b ) gene expression. Total RNA was extracted followed by reverse transcription and real-time PCR analysis of cytokine and chemokine gene expression using GAPDH as an internal control. The baseline gene expression level was set at 1. c – g Different innate immune cell levels in RF and adjuvant-treated skin. Skin was digested in collagenase D and dispase to prepare single-cell suspensions. Cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis of levels of different cell types: neutrophils ( c ), monocytes ( d ), macrophages ( e ), eosinophils ( f ), and mDCs ( g ) (Supplementary Fig. 2). n = 4. Student’s t -test was used to compare differences between groups at 48 and 96 h. *, p

    Techniques Used: Mouse Assay, Injection, Expressing, Real-time Polymerase Chain Reaction, Staining, Fluorescence, Flow Cytometry, Cytometry

    RF increases antigen uptake of DCs in skin. Lateral back skin of C57BL/6 mice was exposed to RF or sham treatment followed by ID injection of 2 µg AF647-OVA into RF or sham-treated skin. ID injection of PBS served as control. Skin was harvested 24 h later and digested in collagenase D and dispase to prepare single-cell suspensions. Skin cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis. a Live cells were gated and plotted based on CD11c and MHC II expression. CD11c + MHC II + cells were gated and plotted based on Langerin expression. b Langerin + cells (gate 1) were plotted based on CD11b and CD103 expression into Langerin + CD11b − CD103 + (I) and Langerin + CD11b + CD103 − subsets (II), whereas Langerin − cells (gate 2) were plotted based on CD11b expression into Langerin − CD11b + (III) and Langerin − CD11b − subsets (IV). c Percentage of AF647 + cells was analyzed for each DC subset. n = 4 for PBS control and 6 for sham and RF groups. Student’s t -test was used to compare differences between RF and sham groups. * p
    Figure Legend Snippet: RF increases antigen uptake of DCs in skin. Lateral back skin of C57BL/6 mice was exposed to RF or sham treatment followed by ID injection of 2 µg AF647-OVA into RF or sham-treated skin. ID injection of PBS served as control. Skin was harvested 24 h later and digested in collagenase D and dispase to prepare single-cell suspensions. Skin cells were then stained with fluorescence-conjugated antibodies followed by flow cytometry analysis. a Live cells were gated and plotted based on CD11c and MHC II expression. CD11c + MHC II + cells were gated and plotted based on Langerin expression. b Langerin + cells (gate 1) were plotted based on CD11b and CD103 expression into Langerin + CD11b − CD103 + (I) and Langerin + CD11b + CD103 − subsets (II), whereas Langerin − cells (gate 2) were plotted based on CD11b expression into Langerin − CD11b + (III) and Langerin − CD11b − subsets (IV). c Percentage of AF647 + cells was analyzed for each DC subset. n = 4 for PBS control and 6 for sham and RF groups. Student’s t -test was used to compare differences between RF and sham groups. * p

    Techniques Used: Mouse Assay, Injection, Staining, Fluorescence, Flow Cytometry, Cytometry, Expressing

    16) Product Images from "Assessment of the innate and adaptive immune system in proliferative vitreoretinopathy"

    Article Title: Assessment of the innate and adaptive immune system in proliferative vitreoretinopathy

    Journal: Eye

    doi: 10.1038/eye.2012.52

    Early inflammatory infiltration occurred at the same speed in WT and KO mice. (a) H E staining of the retina after dispase or saline injection. (b) H E staining of the aqueous angle. Arrows indicate neutrophils. (c) Immunofluorescence analysis showing the absence of T cells (CD3 + ), macrophages (F4/80 + ), and NK cells (CD56 + ) 48 h after dispase injection.
    Figure Legend Snippet: Early inflammatory infiltration occurred at the same speed in WT and KO mice. (a) H E staining of the retina after dispase or saline injection. (b) H E staining of the aqueous angle. Arrows indicate neutrophils. (c) Immunofluorescence analysis showing the absence of T cells (CD3 + ), macrophages (F4/80 + ), and NK cells (CD56 + ) 48 h after dispase injection.

    Techniques Used: Mouse Assay, Staining, Injection, Immunofluorescence

    Detection of OVA-specific T cells in PVR lesions at 48 and 72 h time point. OVA-specific, OT II TCR-transgenic T cells were labeled with CFSE and injected i.v. into congenic Rag-1 KO mice that received a simultaneous intraocular dispase injection. The mice were killed 12, 24, 48, 72, and 96 h later and cryosections of their eyes were studied for the presence of green-fluorescent (CFSE positive) cells. Hoechst 33342 dye was used to stain nuclei of the tissue blue. Anti-CD3 staining is show in red.
    Figure Legend Snippet: Detection of OVA-specific T cells in PVR lesions at 48 and 72 h time point. OVA-specific, OT II TCR-transgenic T cells were labeled with CFSE and injected i.v. into congenic Rag-1 KO mice that received a simultaneous intraocular dispase injection. The mice were killed 12, 24, 48, 72, and 96 h later and cryosections of their eyes were studied for the presence of green-fluorescent (CFSE positive) cells. Hoechst 33342 dye was used to stain nuclei of the tissue blue. Anti-CD3 staining is show in red.

    Techniques Used: Transgenic Assay, Labeling, Injection, Mouse Assay, Staining

    Rag-1 KO mice develop all cardinal features of PVR. Representative PVR manifestations are shown for a total of 45 Rag-1 KO mice studied 2–8 weeks after injection of dispase. (a) Traction membrane in dispase-injected eye as indicated by arrow. (b) Proliferative membrane in vitreal cavity and retinal detachment as indicated by arrow. (c) Immunofluorescence staining for α -SMA (c-1), GFAP (c-2), GS (c-5), and RPE-65 (c-6) in the epiretinal membrane (EM), and Hoechst 33342 staining to mark cell nuclei with the EM (c-3 and c-7). c-4 is the merged picture of c-1–c-3, and c-8 is the merged picture of c-5–c-7 (triple staining).
    Figure Legend Snippet: Rag-1 KO mice develop all cardinal features of PVR. Representative PVR manifestations are shown for a total of 45 Rag-1 KO mice studied 2–8 weeks after injection of dispase. (a) Traction membrane in dispase-injected eye as indicated by arrow. (b) Proliferative membrane in vitreal cavity and retinal detachment as indicated by arrow. (c) Immunofluorescence staining for α -SMA (c-1), GFAP (c-2), GS (c-5), and RPE-65 (c-6) in the epiretinal membrane (EM), and Hoechst 33342 staining to mark cell nuclei with the EM (c-3 and c-7). c-4 is the merged picture of c-1–c-3, and c-8 is the merged picture of c-5–c-7 (triple staining).

    Techniques Used: Mouse Assay, Injection, Immunofluorescence, Staining

    Development of clinical PVR in Rag-1 KO and WT mice after dispase injection. A total of 10 mice in each group were injected intraocularly with 0.2 U/ μ l dispase. At the time points specified, the eyes were examined for: (a) macroscopic presentation of PVR symptoms; and (b) histological evidence for PVR as established on H E-stained sections. (c) Skot ERGs recording α - and β -wave amplitudes. The contralateral untreated eyes were used as controls. The data in both panels represent the cumulative results obtained on 25 Rag-1 KO mice after dispase injection.
    Figure Legend Snippet: Development of clinical PVR in Rag-1 KO and WT mice after dispase injection. A total of 10 mice in each group were injected intraocularly with 0.2 U/ μ l dispase. At the time points specified, the eyes were examined for: (a) macroscopic presentation of PVR symptoms; and (b) histological evidence for PVR as established on H E-stained sections. (c) Skot ERGs recording α - and β -wave amplitudes. The contralateral untreated eyes were used as controls. The data in both panels represent the cumulative results obtained on 25 Rag-1 KO mice after dispase injection.

    Techniques Used: Mouse Assay, Injection, Staining

    17) Product Images from "Ocular pathogenesis and immune reaction after intravitreal dispase injection in mice"

    Article Title: Ocular pathogenesis and immune reaction after intravitreal dispase injection in mice

    Journal: Molecular Vision

    doi:

    Immunofluorescence analysis showed no cluster of differentiation (CD)3+ labeled T cells, F4/80+ labeled macrophages, or CD56+ labeled natural killer (NK) cells involved in dispase-injected eyes at 48 h time point ( A , B ). Bluish cells stained with Hoechst 33342 (scale bar 100 μm).
    Figure Legend Snippet: Immunofluorescence analysis showed no cluster of differentiation (CD)3+ labeled T cells, F4/80+ labeled macrophages, or CD56+ labeled natural killer (NK) cells involved in dispase-injected eyes at 48 h time point ( A , B ). Bluish cells stained with Hoechst 33342 (scale bar 100 μm).

    Techniques Used: Immunofluorescence, Labeling, Injection, Staining

    Intracellular staining at the 4-week time point in single T cell suspensions from dispase-injected PVR-model mouse spleens. A : IFN-γ+/CD4+T cells 0.061% of the total CD4+T cells. B : IFN-γ+/CD8+T cells 0.23% of the total CD8+T cells.
    Figure Legend Snippet: Intracellular staining at the 4-week time point in single T cell suspensions from dispase-injected PVR-model mouse spleens. A : IFN-γ+/CD4+T cells 0.061% of the total CD4+T cells. B : IFN-γ+/CD8+T cells 0.23% of the total CD8+T cells.

    Techniques Used: Staining, Injection

    Dispase-injected mice developed cardinal features of proliferative vitreoretinopathy (PVR). A : Ocular fundi of dispase-injected eye and saline-injected control eye. Arrow shows PVR membranes. B : Hematoxylin and eosin (H E) staining of dispase-injected PVR eye and saline-injected control eye. Arrow shows proliferative epiretinal membranes (EM). C : PVR percentages at 1, 2, 4, 6, and 8-week time points after dispase injection.
    Figure Legend Snippet: Dispase-injected mice developed cardinal features of proliferative vitreoretinopathy (PVR). A : Ocular fundi of dispase-injected eye and saline-injected control eye. Arrow shows PVR membranes. B : Hematoxylin and eosin (H E) staining of dispase-injected PVR eye and saline-injected control eye. Arrow shows proliferative epiretinal membranes (EM). C : PVR percentages at 1, 2, 4, 6, and 8-week time points after dispase injection.

    Techniques Used: Injection, Mouse Assay, Staining

    Electroretinograms (ERG) of the dispase-injected proliferative vitreoretinopathy (PVR) model eyes and saline-injected control eyes. Scotopic ERGs showed significant decreases in b-wave amplitudes, but no decreases in a-wave amplitudes of dispase-injected PVR eyes compared with saline-injected control eyes.
    Figure Legend Snippet: Electroretinograms (ERG) of the dispase-injected proliferative vitreoretinopathy (PVR) model eyes and saline-injected control eyes. Scotopic ERGs showed significant decreases in b-wave amplitudes, but no decreases in a-wave amplitudes of dispase-injected PVR eyes compared with saline-injected control eyes.

    Techniques Used: Injection

    Early inflammatory infiltration profile (0–5 day time points) in diapase-injected and saline-injected control mice. Panels A - G shows inflammatory infiltration profile 0–5 days. Panel H shows a control. Neutrophils appeared from hour 8 ( C ) to hour 48 ( F ) in the anterior chamber of dispase-injected eyes but not saline-injected eyes (hematoxylin and eosin [H E] staining, scale bar 100 μm). All the arrows of panels C - F shows neutrophils.
    Figure Legend Snippet: Early inflammatory infiltration profile (0–5 day time points) in diapase-injected and saline-injected control mice. Panels A - G shows inflammatory infiltration profile 0–5 days. Panel H shows a control. Neutrophils appeared from hour 8 ( C ) to hour 48 ( F ) in the anterior chamber of dispase-injected eyes but not saline-injected eyes (hematoxylin and eosin [H E] staining, scale bar 100 μm). All the arrows of panels C - F shows neutrophils.

    Techniques Used: Injection, Mouse Assay, Staining

    Enzyme-Linked ImmunoSpot (ELISpot) assay of interferon (INF) -γ in single T cell suspensions from dispase-injected and saline-injected mouse spleens. A : Compared with positive control group (stimulated with phytohemagglutinin, PHA), IFN-γ values were only detected at 4-week and 8-week time points in dispase-injected mice, and not detected at any time point in saline-injected mice. B : In the graph of counting statistics, there were only 25.5 points at the 4-week time point, and 2.5 points in the 8-week for dispase-injected mice.
    Figure Legend Snippet: Enzyme-Linked ImmunoSpot (ELISpot) assay of interferon (INF) -γ in single T cell suspensions from dispase-injected and saline-injected mouse spleens. A : Compared with positive control group (stimulated with phytohemagglutinin, PHA), IFN-γ values were only detected at 4-week and 8-week time points in dispase-injected mice, and not detected at any time point in saline-injected mice. B : In the graph of counting statistics, there were only 25.5 points at the 4-week time point, and 2.5 points in the 8-week for dispase-injected mice.

    Techniques Used: Enzyme-linked Immunospot, Injection, Positive Control, Mouse Assay

    18) Product Images from "The inhibitory effect of small interference RNA protein kinase C-alpha on the experimental proliferative vitreoretinopathy induced by dispase in mice"

    Article Title: The inhibitory effect of small interference RNA protein kinase C-alpha on the experimental proliferative vitreoretinopathy induced by dispase in mice

    Journal: International Journal of Nanomedicine

    doi: 10.2147/IJN.S37635

    PKCα protein changes after siRNA-PKCα injection. Notes: Western blot analysis shows that PKCα decreased compared to those from the dispase-injected and control groups (ANOVA, * P = 0.00220
    Figure Legend Snippet: PKCα protein changes after siRNA-PKCα injection. Notes: Western blot analysis shows that PKCα decreased compared to those from the dispase-injected and control groups (ANOVA, * P = 0.00220

    Techniques Used: Injection, Western Blot

    RT-PCR analysis after injection of 1500 nM siRNA-PKCα. Notes: PKCα messenger RNA was significantly down regulated following siRNA-PKCα injection when compared with those that were dispase-injected or in the control groups (ANOVA, ** P = 0.00018
    Figure Legend Snippet: RT-PCR analysis after injection of 1500 nM siRNA-PKCα. Notes: PKCα messenger RNA was significantly down regulated following siRNA-PKCα injection when compared with those that were dispase-injected or in the control groups (ANOVA, ** P = 0.00018

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Injection

    19) Product Images from "Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin"

    Article Title: Nonmuscle myosin IIA is involved in recruitment of apical junction components through activation of α-catenin

    Journal: Biology Open

    doi: 10.1242/bio.031369

    E-cadherin–α-catenin chimeric molecules restore cell–cell adhesion and junction assembly. (A) Schematic representation of E-cadherin and its four derivatives. E-cadherin associates with catenins (α-cat and β-cat). ELA is a mutant E-cadherin in which two leucine residues at positions 587 and 588 were replaced with alanine residues. This substitution improves the cell-surface localization of E-cadherin. ELAαC is an ELA–α-catenin chimeric protein consisting of (a) the entire extracellular and transmembrane domains of E-cadherin, as well as the first 80 amino acids of its cytoplasmic domain, excluding the region required for β-catenin–binding, and (b) α-catenin regions encompassing amino acids 612–906 (see Fig. 5 A). EWALAαC has an additional mutation of the Trp2 to alanine (W2A) in the E-cadherin extracellular domain, which abolishes lateral dimerization and the adhesive interactions of E-cadherin. ELAαM is another ELA–α-catenin chimeric protein containing α-catenin regions encompassing amino acids 157–381, which include the region necessary for association with vinculin. All constructs were tagged with HA. (B) Immunoblot detection of ELA, ELAαC, EWALAαC, and ELAαM chimeras expressed in GFP–IIA/IIAKO cells. Cells expressing the respective chimeras were cultured in the presence or absence of Dox for 4 days, and cell lysates prepared were analyzed. Blots were stained with anti-HA, anti-GFP, or anti-vinculin antibodies. Vinculin was used as a loading control. (C) Morphology of cells expressing the chimeras. Cells were cultured in the presence of Dox to suppress GFP–IIA expression, and then observed using a phase-contrast microscope. (D) Cells cultured in the presence of Dox were incubated with Dispase, and then detached cells were subjected to dissociation assays and quantified. Values represent the mean±s.e.; n =at least 3 times. * P
    Figure Legend Snippet: E-cadherin–α-catenin chimeric molecules restore cell–cell adhesion and junction assembly. (A) Schematic representation of E-cadherin and its four derivatives. E-cadherin associates with catenins (α-cat and β-cat). ELA is a mutant E-cadherin in which two leucine residues at positions 587 and 588 were replaced with alanine residues. This substitution improves the cell-surface localization of E-cadherin. ELAαC is an ELA–α-catenin chimeric protein consisting of (a) the entire extracellular and transmembrane domains of E-cadherin, as well as the first 80 amino acids of its cytoplasmic domain, excluding the region required for β-catenin–binding, and (b) α-catenin regions encompassing amino acids 612–906 (see Fig. 5 A). EWALAαC has an additional mutation of the Trp2 to alanine (W2A) in the E-cadherin extracellular domain, which abolishes lateral dimerization and the adhesive interactions of E-cadherin. ELAαM is another ELA–α-catenin chimeric protein containing α-catenin regions encompassing amino acids 157–381, which include the region necessary for association with vinculin. All constructs were tagged with HA. (B) Immunoblot detection of ELA, ELAαC, EWALAαC, and ELAαM chimeras expressed in GFP–IIA/IIAKO cells. Cells expressing the respective chimeras were cultured in the presence or absence of Dox for 4 days, and cell lysates prepared were analyzed. Blots were stained with anti-HA, anti-GFP, or anti-vinculin antibodies. Vinculin was used as a loading control. (C) Morphology of cells expressing the chimeras. Cells were cultured in the presence of Dox to suppress GFP–IIA expression, and then observed using a phase-contrast microscope. (D) Cells cultured in the presence of Dox were incubated with Dispase, and then detached cells were subjected to dissociation assays and quantified. Values represent the mean±s.e.; n =at least 3 times. * P

    Techniques Used: Mutagenesis, Binding Assay, Construct, Expressing, Cell Culture, Staining, Microscopy, Incubation

    The α-catenin construct α1–381 requires vinculin for its ability to rescue junction formation. Using the CRISPR/Cas9 system, the vinculin gene was ablated in α1–381/GFP–IIA/IIAKO cells, yielding α1–381/GFP–IIA/IIAKO-vincKO double-knockout cells. (A) Immunoblot detection of vinculin (vinc) and actin. α1–381/GFP–IIA/IIAKO cells with wild-type vinculin (vincWT) were used as a positive control, and actin was used as a loading control. (B) Genomic sequencing revealed a mutation in the target region of vinculin gene that induced a frame-shift. All 13 independent clones sequenced harbored the same insertion of 1 bp. (C) Morphology of α1–381/GFP–IIA/IIAKO-vincKO cells. Cells cultured in the presence of Dox were observed using a phase-contrast microscope. (D) Cells cultured in the presence of Dox were incubated with Dispase, and then detached cells were subjected to dissociation assays and quantified. Values represent the mean±s.e.; n =at least 3 times. * P
    Figure Legend Snippet: The α-catenin construct α1–381 requires vinculin for its ability to rescue junction formation. Using the CRISPR/Cas9 system, the vinculin gene was ablated in α1–381/GFP–IIA/IIAKO cells, yielding α1–381/GFP–IIA/IIAKO-vincKO double-knockout cells. (A) Immunoblot detection of vinculin (vinc) and actin. α1–381/GFP–IIA/IIAKO cells with wild-type vinculin (vincWT) were used as a positive control, and actin was used as a loading control. (B) Genomic sequencing revealed a mutation in the target region of vinculin gene that induced a frame-shift. All 13 independent clones sequenced harbored the same insertion of 1 bp. (C) Morphology of α1–381/GFP–IIA/IIAKO-vincKO cells. Cells cultured in the presence of Dox were observed using a phase-contrast microscope. (D) Cells cultured in the presence of Dox were incubated with Dispase, and then detached cells were subjected to dissociation assays and quantified. Values represent the mean±s.e.; n =at least 3 times. * P

    Techniques Used: Construct, CRISPR, Double Knockout, Positive Control, Genomic Sequencing, Mutagenesis, Clone Assay, Cell Culture, Microscopy, Incubation

    Expression of α-catenin deletion constructs in IIAKO cells induces junction assembly and restores cell–cell adhesion. (A) Schematic representation of α-catenin (top) and mutant derivatives (bottom). The N-terminal (N) domain binds to β-catenin. The middle (M) domain comprises three regions, MI, MII, and MIII. MI contains binding sites for vinculin, and MII and MIII are involved in regulation of force-dependent vinculin binding. The C-terminal (C) domain binds actin. α1–302 does not contain the vinculin- and actin-binding sites. α1–381 is a construct in which the carboxy-terminal 525 residues have been truncated, thereby removing the actin-binding site but retaining the vinculin-binding site. αΔ203–612 has an internal deletion of residues 203–612, and therefore lacks the vinculin-binding site but retains the actin-binding site. All constructs were tagged with HA. (B) Immunoblot detection of α-catenin polypeptides. GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612 were cultured in the presence or absence of Dox for 4 days, and then analyzed. The blots were probed with anti-HA, anti-GFP, and anti-vinculin antibodies. (C) Immunofluorescence staining with anti-HA revealed that these constructs were localized at cell–cell contact sites of cells cultured in the absence of Dox. (D) Morphology of GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612. Cells cultured in the presence of Dox to suppress GFP–IIA expression, and were observed using a phase contrast microscope. (E) Dissociation assays. Cells were incubated with Dispase, and then detached cells were subjected to mechanical stress by pipetting and quantified. Values represent the mean±s.e.; n =at least 3 times. * P
    Figure Legend Snippet: Expression of α-catenin deletion constructs in IIAKO cells induces junction assembly and restores cell–cell adhesion. (A) Schematic representation of α-catenin (top) and mutant derivatives (bottom). The N-terminal (N) domain binds to β-catenin. The middle (M) domain comprises three regions, MI, MII, and MIII. MI contains binding sites for vinculin, and MII and MIII are involved in regulation of force-dependent vinculin binding. The C-terminal (C) domain binds actin. α1–302 does not contain the vinculin- and actin-binding sites. α1–381 is a construct in which the carboxy-terminal 525 residues have been truncated, thereby removing the actin-binding site but retaining the vinculin-binding site. αΔ203–612 has an internal deletion of residues 203–612, and therefore lacks the vinculin-binding site but retains the actin-binding site. All constructs were tagged with HA. (B) Immunoblot detection of α-catenin polypeptides. GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612 were cultured in the presence or absence of Dox for 4 days, and then analyzed. The blots were probed with anti-HA, anti-GFP, and anti-vinculin antibodies. (C) Immunofluorescence staining with anti-HA revealed that these constructs were localized at cell–cell contact sites of cells cultured in the absence of Dox. (D) Morphology of GFP–IIA/IIAKO cells expressing α1–302, α1–381, or αΔ203–612. Cells cultured in the presence of Dox to suppress GFP–IIA expression, and were observed using a phase contrast microscope. (E) Dissociation assays. Cells were incubated with Dispase, and then detached cells were subjected to mechanical stress by pipetting and quantified. Values represent the mean±s.e.; n =at least 3 times. * P

    Techniques Used: Expressing, Construct, Mutagenesis, Binding Assay, Cell Culture, Immunofluorescence, Staining, Microscopy, Incubation

    20) Product Images from "DEC-205lo Langerinlo neonatal Langerhans' cells preferentially utilize a wortmannin-sensitive, fluid-phase pathway to internalize exogenous antigen"

    Article Title: DEC-205lo Langerinlo neonatal Langerhans' cells preferentially utilize a wortmannin-sensitive, fluid-phase pathway to internalize exogenous antigen

    Journal: Immunology

    doi: 10.1111/j.1365-2567.2003.01759.x

    Identification of Langerhans' cells (LC) in epidermal suspensions. A gating strategy is shown for the identification of LC in epidermal suspensions. Epidermal suspensions were prepared from murine epidermis by digestion with dispase at 4 days (top panel), 7 days (second top panel), 14 days (second bottom panel) and 6 weeks (bottom panel) and then stained with anti-major histocompatibility complex class II (MHC class II). To select for viable cells, a gate was first drawn around 7-aminoactinomycin D-negative (7-AAD − ) cells (data not shown). LC are represented by the discrete population of viable MHC class II + cells (data are representative of three or more experiments).
    Figure Legend Snippet: Identification of Langerhans' cells (LC) in epidermal suspensions. A gating strategy is shown for the identification of LC in epidermal suspensions. Epidermal suspensions were prepared from murine epidermis by digestion with dispase at 4 days (top panel), 7 days (second top panel), 14 days (second bottom panel) and 6 weeks (bottom panel) and then stained with anti-major histocompatibility complex class II (MHC class II). To select for viable cells, a gate was first drawn around 7-aminoactinomycin D-negative (7-AAD − ) cells (data not shown). LC are represented by the discrete population of viable MHC class II + cells (data are representative of three or more experiments).

    Techniques Used: Staining

    21) Product Images from "Impact of the Stroma on the Biological Characteristics of the Parenchyma in Oral Squamous Cell Carcinoma"

    Article Title: Impact of the Stroma on the Biological Characteristics of the Parenchyma in Oral Squamous Cell Carcinoma

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms21207714

    Isolation of various stromal cells. Pieces of fresh oral squamous carcinoma tissue (1 mm 3 ) were washed several times with Alpha-MEM and then minced. These tissues were treated with Alpha-MEM containing 1 mg/mL collagenase II and Dispase for 2 h at 37 °C with shaking (200 rpm). The released cells were centrifuged for 5 min at 1000 rpm, suspended in Alpha-MEM containing 10% FBS, filtered using a Cell strainer, plated in a tissue culture flask, and incubated at 37 °C in 5% CO 2 . One week later, stromal cells were obtained following treatment with Accutase based on the different adhesion of epithelial and stromal cells.
    Figure Legend Snippet: Isolation of various stromal cells. Pieces of fresh oral squamous carcinoma tissue (1 mm 3 ) were washed several times with Alpha-MEM and then minced. These tissues were treated with Alpha-MEM containing 1 mg/mL collagenase II and Dispase for 2 h at 37 °C with shaking (200 rpm). The released cells were centrifuged for 5 min at 1000 rpm, suspended in Alpha-MEM containing 10% FBS, filtered using a Cell strainer, plated in a tissue culture flask, and incubated at 37 °C in 5% CO 2 . One week later, stromal cells were obtained following treatment with Accutase based on the different adhesion of epithelial and stromal cells.

    Techniques Used: Isolation, Incubation

    22) Product Images from "Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells"

    Article Title: Conditional requirement for the Flk-1 receptor in the in vitro generation of early hematopoietic cells

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

    doi:

    β-gal and CD31 expression in flk-1 (+/−) and flk-1 (−/−) EBs. Photomicrographs of day 8 after dispase attached EB cultures stained for β-gal activity with FDG ( A and C ) and CD31 expression stained with phycoerythrin-conjugated anti-CD31 ( B and D ). (×4.) flk-1 +/− EBs form a vascular endothelial network expressing β-gal ( A ) and CD31 ( B ). Although flk-1 −/− ES cells differentiate into β-gal-positive cells ( C ), few (arrows) also express CD31 ( D ), and none exhibit an endothelial-like morphology.
    Figure Legend Snippet: β-gal and CD31 expression in flk-1 (+/−) and flk-1 (−/−) EBs. Photomicrographs of day 8 after dispase attached EB cultures stained for β-gal activity with FDG ( A and C ) and CD31 expression stained with phycoerythrin-conjugated anti-CD31 ( B and D ). (×4.) flk-1 +/− EBs form a vascular endothelial network expressing β-gal ( A ) and CD31 ( B ). Although flk-1 −/− ES cells differentiate into β-gal-positive cells ( C ), few (arrows) also express CD31 ( D ), and none exhibit an endothelial-like morphology.

    Techniques Used: Expressing, Staining, Activity Assay

    23) Product Images from "A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis"

    Article Title: A mesenchyme-free culture system to elucidate the mechanism of otic vesicle morphogenesis

    Journal: Journal of Anatomy

    doi: 10.1111/j.0021-8782.2004.00335.x

    Culture method. Hindbrain regions of E10.5 or 11.5 mouse embryos are isolated using microscissors, and incubated in 1 U mL −1 dispase. Otic vesicles are isolated using fine tungsten needles and embedded in a drop of Matrigel. The drop is allowed to gel at 37 °C in a humidified atmosphere for 30 min, then culture medium is added to the dish and culture maintained for up to 72 h.
    Figure Legend Snippet: Culture method. Hindbrain regions of E10.5 or 11.5 mouse embryos are isolated using microscissors, and incubated in 1 U mL −1 dispase. Otic vesicles are isolated using fine tungsten needles and embedded in a drop of Matrigel. The drop is allowed to gel at 37 °C in a humidified atmosphere for 30 min, then culture medium is added to the dish and culture maintained for up to 72 h.

    Techniques Used: Isolation, Incubation

    24) Product Images from "Whole lung irradiation results in pulmonary macrophage alterations that are subpopulation and strain specific"

    Article Title: Whole lung irradiation results in pulmonary macrophage alterations that are subpopulation and strain specific

    Journal: Radiation research

    doi: 10.1667/RR14178.1

    Gating strategy and flow cytometric characterization of pulmonary macrophage populations. 1A: Lungs of C3H and C57 mice were dispase digested and enriched for CD45+ cells using MACS 3–26 wk following 12.5 Gy whole lung irradiation. Cells were
    Figure Legend Snippet: Gating strategy and flow cytometric characterization of pulmonary macrophage populations. 1A: Lungs of C3H and C57 mice were dispase digested and enriched for CD45+ cells using MACS 3–26 wk following 12.5 Gy whole lung irradiation. Cells were

    Techniques Used: Flow Cytometry, Mouse Assay, Magnetic Cell Separation, Irradiation

    25) Product Images from "Enzyme-free Passage of Human Pluripotent Stem Cells by Controlling Divalent Cations"

    Article Title: Enzyme-free Passage of Human Pluripotent Stem Cells by Controlling Divalent Cations

    Journal: Scientific Reports

    doi: 10.1038/srep04646

    Effects of dissociation without divalent cation and with dispase. (a–c): Comparison between the hiPSCs (253G1) detached and dissociated by PBS −/− (−/−) and those by PBS ca/− (Ca). (ab): Micrograph (a) and FCM analysis (b) of apoptosis marker annexin V-FITC after four hours floating culture (RI: with 5 μM ROCK inhibitor) following detachment and dissociation. The cells were stained by annexin V-FITC (green), propidium iodide (red: late apoptosis or necrosis marker), and Hoechst 33342 (blue: nuclei marker). (c): The number of cells remaining after 3 days culture in ESF9a on fibronectin-coated 10-cm dishes (the initial cell number was 1.2 × 10 5 cells/10-cm dish, mean ± SE, n = 5, t-test). (d–g): Effects of dispase in PBS ca/− for detaching and dissociating cells. (d): Mean cell clump size detached from the plates using PBS ca/− (Ca) and 1 U/ml dispase in PBS ca/− (Ca + Ds). (ef): Micrograph (e) and FCM analysis (f) of annexin V-FITC after four hours floating culture following detachment and dissociation by PBS ca/− or 1 U/ml dispase in PBS ca/− . The staining were the same as (a). (g): Reattachment efficiency. The cells were digested with 0–2 U/ml dispase in PBS ca/− and were plated with ESF9a medium including 5 μM ROCK inhibitor. The numbers of cells were estimated using calcein-AM 1 day after plating and normalized against the PBS ca/− results (0 U/ml dispase). The mean value at 1 U/ml dispase was smaller than 1 ( P
    Figure Legend Snippet: Effects of dissociation without divalent cation and with dispase. (a–c): Comparison between the hiPSCs (253G1) detached and dissociated by PBS −/− (−/−) and those by PBS ca/− (Ca). (ab): Micrograph (a) and FCM analysis (b) of apoptosis marker annexin V-FITC after four hours floating culture (RI: with 5 μM ROCK inhibitor) following detachment and dissociation. The cells were stained by annexin V-FITC (green), propidium iodide (red: late apoptosis or necrosis marker), and Hoechst 33342 (blue: nuclei marker). (c): The number of cells remaining after 3 days culture in ESF9a on fibronectin-coated 10-cm dishes (the initial cell number was 1.2 × 10 5 cells/10-cm dish, mean ± SE, n = 5, t-test). (d–g): Effects of dispase in PBS ca/− for detaching and dissociating cells. (d): Mean cell clump size detached from the plates using PBS ca/− (Ca) and 1 U/ml dispase in PBS ca/− (Ca + Ds). (ef): Micrograph (e) and FCM analysis (f) of annexin V-FITC after four hours floating culture following detachment and dissociation by PBS ca/− or 1 U/ml dispase in PBS ca/− . The staining were the same as (a). (g): Reattachment efficiency. The cells were digested with 0–2 U/ml dispase in PBS ca/− and were plated with ESF9a medium including 5 μM ROCK inhibitor. The numbers of cells were estimated using calcein-AM 1 day after plating and normalized against the PBS ca/− results (0 U/ml dispase). The mean value at 1 U/ml dispase was smaller than 1 ( P

    Techniques Used: Marker, Staining

    26) Product Images from "Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis"

    Article Title: Desmoglein-1 regulates esophageal epithelial barrier function and immune responses in eosinophilic esophagitis

    Journal: Mucosal immunology

    doi: 10.1038/mi.2013.90

    Loss of DSG1 reduces esophageal epithelial cell adhesion qPCR analysis of DSG1 (A) and DSG3 (B) in ALI-differentiated EPC2 cells stably transduced with non-silencing control (NSC) or DSG1 shRNA. H E-stained sections from stably transduced cells differentiated at the ALI (C). H E-stained sections from EPC2 cells exposed to the air interface and treated with 10 μg/mL ETA (WT) or the S195A inactive mutant for 24 h (D). Arrows (C–D) indicate cell separation within the suprabasal epithelium. Cytospins from NSC or DSG1 shRNA-transduced EPC2 cells following dispase adhesion assays (E) and quantification of dissociated cell clusters are shown (F). Images in (C–E) are representative of 4–5 experiments performed in duplicate. Data in (A–B) and (E) are from 3 experiments performed in duplicate and are represented as the mean + SEM: NS (not significant), * p
    Figure Legend Snippet: Loss of DSG1 reduces esophageal epithelial cell adhesion qPCR analysis of DSG1 (A) and DSG3 (B) in ALI-differentiated EPC2 cells stably transduced with non-silencing control (NSC) or DSG1 shRNA. H E-stained sections from stably transduced cells differentiated at the ALI (C). H E-stained sections from EPC2 cells exposed to the air interface and treated with 10 μg/mL ETA (WT) or the S195A inactive mutant for 24 h (D). Arrows (C–D) indicate cell separation within the suprabasal epithelium. Cytospins from NSC or DSG1 shRNA-transduced EPC2 cells following dispase adhesion assays (E) and quantification of dissociated cell clusters are shown (F). Images in (C–E) are representative of 4–5 experiments performed in duplicate. Data in (A–B) and (E) are from 3 experiments performed in duplicate and are represented as the mean + SEM: NS (not significant), * p

    Techniques Used: Real-time Polymerase Chain Reaction, Stable Transfection, Transduction, shRNA, Staining, Mutagenesis

    27) Product Images from "Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines"

    Article Title: Specific antibody-receptor interactions trigger InlAB-independent uptake of listeria monocytogenes into tumor cell lines

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-11-163

    Antibody-mediated targeting of uncoated (-mAb), Cetuximab- or Trastuzumab- coated Lm-spa + after antibody crosslinking in xenografted mouse tumor models . In seven Balb/c SCID mice per group 4T1-HER2 tumors were induced and 14 days later the mice were infected with 1 × 10 8 CFU of differently coated Lm-spa + . 24 h later mice were sacrificed and tumors, liver and spleen excised aseptically. Tumors were digested with DNAse and Dispase to obtain single cell suspensions which were plated in serial dilutions without ( a ) and with gentamicin treatment ( b ) to determine total and intracellular bacterial counts, respectively. Depicted is the bacterial count per cell in the cell suspension. Liver ( c ) and spleen ( d ) were homogenized and plated in serial dilutions.
    Figure Legend Snippet: Antibody-mediated targeting of uncoated (-mAb), Cetuximab- or Trastuzumab- coated Lm-spa + after antibody crosslinking in xenografted mouse tumor models . In seven Balb/c SCID mice per group 4T1-HER2 tumors were induced and 14 days later the mice were infected with 1 × 10 8 CFU of differently coated Lm-spa + . 24 h later mice were sacrificed and tumors, liver and spleen excised aseptically. Tumors were digested with DNAse and Dispase to obtain single cell suspensions which were plated in serial dilutions without ( a ) and with gentamicin treatment ( b ) to determine total and intracellular bacterial counts, respectively. Depicted is the bacterial count per cell in the cell suspension. Liver ( c ) and spleen ( d ) were homogenized and plated in serial dilutions.

    Techniques Used: Mouse Assay, Infection

    Related Articles

    Modification:

    Article Title: Entry of Herpes Simplex Virus 1 into Epidermis and Dermal Fibroblasts Is Independent of the Scavenger Receptor MARCO
    Article Snippet: .. Epidermal sheets were prepared by the removal of the dermis by dispase II treatment and incubated in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX supplement [Life Technologies]), as described previously ( ). .. Primary fibroblasts were isolated from murine dermis of control, MARCO−/− , or MARCO−/− /SR-A1−/− dKO adult mice or from lamina propria of human oral mucosa after 3 h of treatment at 37°C in serum-free DMEM-high glucose-GlutaMAX (Life Technologies) containing 400 U of collagenase I (Worthington)/ml as described previously ( ).

    Isolation:

    Article Title: Strain dependent differences in glucocorticoid-induced bone loss between C57BL/6J and CD-1 mice
    Article Snippet: .. In vitro osteogenensis assay Murine calvarial osteoblasts (OBL) isolated as described previously at day 1 of life by sequential enzymatic digestion with 1 mg/ml Collagenase II (Worthington, Lakewood, USA) and 2 mg/ml Dispase (Invitrogen, Paisley, UK) in PBS were plated directly in 96 well plates at 5 × 10^4 cells/well in control medium (DMEM and 10% FBS) or osteogenic medium (DMEM supplemented with 10% FBS, 1 M β-glycerolphosphate, 50 mg/ml phosphoascorbic acid and 0–10^4 nM Dex). .. For evaluating mineralized calvarial bone nodules on day 6 of culture, cells were stained with 1% alizarin red-S (Sigma-Aldrich, Gillingham, UK).

    Incubation:

    Article Title: Entry of Herpes Simplex Virus 1 into Epidermis and Dermal Fibroblasts Is Independent of the Scavenger Receptor MARCO
    Article Snippet: .. Epidermal sheets were prepared by the removal of the dermis by dispase II treatment and incubated in Dulbecco's modified Eagle's medium (DMEM, high glucose, GlutaMAX supplement [Life Technologies]), as described previously ( ). .. Primary fibroblasts were isolated from murine dermis of control, MARCO−/− , or MARCO−/− /SR-A1−/− dKO adult mice or from lamina propria of human oral mucosa after 3 h of treatment at 37°C in serum-free DMEM-high glucose-GlutaMAX (Life Technologies) containing 400 U of collagenase I (Worthington)/ml as described previously ( ).

    Article Title: Cadherin Cytoplasmic Domains Inhibit the Cell Surface Localization of Endogenous E-Cadherin, Blocking Desmosome and Tight Junction Formation and Inducing Cell Dissociation
    Article Snippet: .. Dissociation assay Cells were washed with PBS and then incubated for 2 h in DMEM supplemented with 10% FCS containing 2.4 U/ml of dispase (Gibco). .. Detached cells were subjected to mechanical stress by pipetting with a 1 ml pipette.

    other:

    Article Title: A New Technique for Primary Hepatocyte Expansion In Vitro
    Article Snippet: Long-term cultures of hepatocytes (35 days after seeding) were treated with collagenase (1 mg/mL; Sigma Chemical Co.) and dispase (1 mg/mL; Gibco).

    In Vitro:

    Article Title: Strain dependent differences in glucocorticoid-induced bone loss between C57BL/6J and CD-1 mice
    Article Snippet: .. In vitro osteogenensis assay Murine calvarial osteoblasts (OBL) isolated as described previously at day 1 of life by sequential enzymatic digestion with 1 mg/ml Collagenase II (Worthington, Lakewood, USA) and 2 mg/ml Dispase (Invitrogen, Paisley, UK) in PBS were plated directly in 96 well plates at 5 × 10^4 cells/well in control medium (DMEM and 10% FBS) or osteogenic medium (DMEM supplemented with 10% FBS, 1 M β-glycerolphosphate, 50 mg/ml phosphoascorbic acid and 0–10^4 nM Dex). .. For evaluating mineralized calvarial bone nodules on day 6 of culture, cells were stained with 1% alizarin red-S (Sigma-Aldrich, Gillingham, UK).

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    Thermo Fisher dispase ii
    Oral mucosal cell sheets from 3 donors harvested by mechanical or enzymatic methods <t>(dispase</t> or collagenase) were analyzed by hematoxylin–phloxine–saffron (HPS) staining (scale bars: 100 μm); expression of p63 was analyzed by immunohistochemistry (scale bar = 100 μm); and laminin-332, β1-integrin, E-cadherin, and type VII collagen levels were viewed by immunofluorescence in green color, cell nuclei are shown in blue color (scale bar = 50 μm).
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    Oral mucosal cell sheets from 3 donors harvested by mechanical or enzymatic methods (dispase or collagenase) were analyzed by hematoxylin–phloxine–saffron (HPS) staining (scale bars: 100 μm); expression of p63 was analyzed by immunohistochemistry (scale bar = 100 μm); and laminin-332, β1-integrin, E-cadherin, and type VII collagen levels were viewed by immunofluorescence in green color, cell nuclei are shown in blue color (scale bar = 50 μm).

    Journal: Cell Transplantation

    Article Title: Preserving Basement Membranes during Detachment of Cultivated Oral Mucosal Epithelial Cell Sheets for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

    doi: 10.1177/0963689717741140

    Figure Lengend Snippet: Oral mucosal cell sheets from 3 donors harvested by mechanical or enzymatic methods (dispase or collagenase) were analyzed by hematoxylin–phloxine–saffron (HPS) staining (scale bars: 100 μm); expression of p63 was analyzed by immunohistochemistry (scale bar = 100 μm); and laminin-332, β1-integrin, E-cadherin, and type VII collagen levels were viewed by immunofluorescence in green color, cell nuclei are shown in blue color (scale bar = 50 μm).

    Article Snippet: The epithelium was separated from the lamina propria using dispase II (Thermo Fisher Scientific, Waltham, MA, USA), 10 mg/mL for 3 h at 4 °C.

    Techniques: Staining, Expressing, Immunohistochemistry, Immunofluorescence

    Impact of Dispase and Liberase/Collagenase D digestion methods on populations of immune cells. (A) Single-cell suspensions were prepared using Liberase TL/Collagenase D (Liberase) or Dispase enzyme cocktails and isolated cells co-stained with antibodies prior to analysis by flow cytometry. Cells were initially gated based on CD45 expression and subsequently gated on CD3 and CD4 expression as shown. CD3 hi CD4 − expression marks dendritic epidermal T cells (DETCs) located in mouse epidermis and CD3 + CD4 + expression is detected on dermal infiltrating T cells. (B) Graph representing the number of events as counted by flow cytometry in a 1 mm 2 area of wound tissue. Note tissue digestion using Dispase leads to an approximate 10-fold increase in total cell numbers; however, the number of CD45 + immune cells was similar regardless of digestion method. (C–F) Histograms show expression of cell surface markers in gated Dispase II digestion (red line) and Liberase TL/Collagenase D digestion (filled light blue) populations. With the exception of CD4, there was no detectable difference in intensity of surface marker staining.

    Journal: Frontiers in Immunology

    Article Title: Quantitative Methods for Measuring Repair Rates and Innate-Immune Cell Responses in Wounded Mouse Skin

    doi: 10.3389/fimmu.2018.00347

    Figure Lengend Snippet: Impact of Dispase and Liberase/Collagenase D digestion methods on populations of immune cells. (A) Single-cell suspensions were prepared using Liberase TL/Collagenase D (Liberase) or Dispase enzyme cocktails and isolated cells co-stained with antibodies prior to analysis by flow cytometry. Cells were initially gated based on CD45 expression and subsequently gated on CD3 and CD4 expression as shown. CD3 hi CD4 − expression marks dendritic epidermal T cells (DETCs) located in mouse epidermis and CD3 + CD4 + expression is detected on dermal infiltrating T cells. (B) Graph representing the number of events as counted by flow cytometry in a 1 mm 2 area of wound tissue. Note tissue digestion using Dispase leads to an approximate 10-fold increase in total cell numbers; however, the number of CD45 + immune cells was similar regardless of digestion method. (C–F) Histograms show expression of cell surface markers in gated Dispase II digestion (red line) and Liberase TL/Collagenase D digestion (filled light blue) populations. With the exception of CD4, there was no detectable difference in intensity of surface marker staining.

    Article Snippet: Tissue was briefly rinsed in 1× PBS and was cut the tissue into 4–5 smaller pieces before placing the pieces in a 1.5-mL Eppendorf tube containing a Liberase TL enzyme cocktail [0.35-mg/mL Liberase TL (Roche, cat. #5401020001), 3-mg/mL Collagenase D (Roche, cat. #11088866001), and 0.1-mg/mL DNase I (Roche, cat. #10104159001)], or Dispase II enzyme cocktail (2 mg/mL, Thermo Fisher 17105-041 and 0.1-mg/mL DNase I) in a volume of 700-µL RPMI-1640 (Thermo Fisher, 31870-025) media and incubate on a block heating shaker at 1,400 rpm, 37°C for 2 h. After incubation, remove undigested debris by filtering the sample through the side of a 70-µm strainer into a new collection Eppendorf tube.

    Techniques: Isolation, Staining, Flow Cytometry, Cytometry, Expressing, Marker

    Oral mucosal cell sheets from 3 donors harvested by mechanical or enzymatic methods (dispase or collagenase) were analyzed by hematoxylin–phloxine–saffron (HPS) staining (scale bars: 100 μm); expression of p63 was analyzed by immunohistochemistry (scale bar = 100 μm); and laminin-332, β1-integrin, E-cadherin, and type VII collagen levels were viewed by immunofluorescence in green color, cell nuclei are shown in blue color (scale bar = 50 μm).

    Journal: Cell Transplantation

    Article Title: Preserving Basement Membranes during Detachment of Cultivated Oral Mucosal Epithelial Cell Sheets for the Treatment of Total Bilateral Limbal Stem Cell Deficiency

    doi: 10.1177/0963689717741140

    Figure Lengend Snippet: Oral mucosal cell sheets from 3 donors harvested by mechanical or enzymatic methods (dispase or collagenase) were analyzed by hematoxylin–phloxine–saffron (HPS) staining (scale bars: 100 μm); expression of p63 was analyzed by immunohistochemistry (scale bar = 100 μm); and laminin-332, β1-integrin, E-cadherin, and type VII collagen levels were viewed by immunofluorescence in green color, cell nuclei are shown in blue color (scale bar = 50 μm).

    Article Snippet: As dispase II at 5 mg/mL is validated in our lab for epidermal cell sheet detachment, we chose to use this concentration and test 2 others obtained by 2-fold serial dilutions 2.5 and 1.25 mg/mL to minimize the potential enzymatic degradation.

    Techniques: Staining, Expressing, Immunohistochemistry, Immunofluorescence