Journal:

Article Title: Identification of Novel Principles of Keratin Filament Network Turnover in Living Cells V⃞

doi: 10.1091/mbc.E03-09-0707

Figure Lengend Snippet: Details of KF network formation in living SW13 cells synthesizing HK8-CFP and HK18-YFP fluorescent hybrids. (A) Epifluorescence microscopy (detection of HK18-YFP) of a characteristic peripheral segment (position of plasma membrane indicated at 3.5 min) highlighting intermediate steps of KF network production. Two KF precursors are marked by color and followed until integration into the filament system. Note the formation of small granules below the cell surface that elongate, fuse, and become part of the KF network. The corresponding movie 5 shows further details. (B) Size changes of a single particle that was tracked from its first detection until integration into the network. For clarity's sake only the pixels associated with that particular particle are shown. Sampling frequency, 30 s. (C) The graph is derived from B, showing a surface view of the altering particle dimensions demonstrating a continuous, nonincremental particle elongation (l) before fusion with the KF network over time (t). (D) 3D reconstruction (voltex presentation) of the HK18-YFP fluorescence as recorded by confocal laser scanning microscopy of a peripheral region revealing spatial characteristics of various intermediates of filament assembly in relationship to intracellular topology (position of cell border demarcated). The volume of the top segment has the dimensions 20 × 20 × 3.5 μm. The bottom cube is a cross section to specifically highlight the increase in diameter of KF precursors only after integration into the peripheral KF network. (E) Plot summarizing the quantitative analysis of 10 time-space diagrams (see, e.g., C) depicting the increase in length (top graph) and diameter (bottom graph). Note the continuous elongation of particles during the entire 20 min in contrast to the increase in diameter that is limited to the first 5 min. Bars, SD. Bars, 1 μm in A and D.

Article Snippet: Cell Lines and cDNA Transfection The following cell lines were used and cultured according to the specifications given by ATCC or the respective references: rat kangaroo kidney PtK2 cells (ATCC CCL-56), Madin-Darby canine kidney (MDCK) cells (clone 20; ATCC CCL-34), bovine kidney-derived MDBK cells (ATCC CCL-22), human mammary adenocarcinoma-derived MCF7 cells (ATCC HTB-22), human hepatocellular carcinoma-derived PLC cells (ATCC CRL8024), human colon carcinoma CaCo-2 cells (ATCC HTB-37), human immortalized HaCaT keratinocytes ( Boukamp et al., 1988 ), and human small-cell carcinoma SW13 cells derived from the adrenal cortex (ATCC CCL-105).

Techniques: Epifluorescence Microscopy, Clinical Proteomics, Membrane, Single Particle, Sampling, Derivative Assay, Fluorescence, Confocal Laser Scanning Microscopy

Journal: Cell

Article Title: Nuclear Proximity of Mtr4 with RNA exosome restricts DNA mutational asymmetry

doi: 10.1016/j.cell.2017.03.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Splenic B cells of TAPtag mice were isolated using CD43 magnetic beads (Miltenyi Biotec) negative selection and cultured in RPMI1640 containing 15% FBS with 20 μg/ml LPS (Sigma-Aldrich) and/or 20 ng/ml IL4 (R&D) for 48~72 hrs.

Techniques: Magnetic Beads, Recombinant, Protease Inhibitor, Modification, Electron Microscopy, Shear, Transgenic Assay, Sequencing, Subcloning, Software

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Peptide–major histocompatibility complex (pMHC) dextramers bind CD8 + T cells substantially better than tetramers when T cell receptor (TCR)–pMHC affinity is low. (a) A schematic showing how pMHC dextramers differ from pMHC tetramers. Dextramers have a dextran backbone and carry more fluorochrome and pMHC per molecule. The fluorochrome/pMHC ratio differs between allophycocyanin (APC) and R-phycoerythrin (PE) dextramers. (a) is available as a separate high-resolution file (Supporting Information ) in the online supplement that accompanies this article. (b) The ILA1 CD8 + T cell clone was stained with PE-conjugated tetramer (right) or dextramer (left) reagents constructed with human leucocyte antigen (HLA)-A2 monomers bearing the wild-type human telomerase reverse transcriptase (hTERT) 540–548 peptide (ILAKFLHWL), variants thereof with defined TCR–pMHC-I affinities as indicated, or the HIV-1 Pol 476–484 peptide (ILKEPVHGV) as an irrelevant control. (c) Human peripheral blood mononuclear cells (PBMCs) (10 6 ) were spiked with clonal ILA1 cells (10 4 ) and stained with the indicated tetramers (bottom) or dextramers (top), the viability dye Aqua and monoclonal antibodies against the surface markers CD3, CD4, CD8, CD14 and CD19. Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells prior to display as CD8 versus tetramer/dextramer. In each column, the red box is positioned on the antigen-specific CD8 + T cell population that stains most brightly to aid visual comparison.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Immunopeptidomics, Staining, Construct, Reverse Transcription, Control, Bioprocessing, Comparison

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Dextramers made with peptide–major histocompatibility complex (pMHC) class II bind CD4 + T cells substantially better than tetramers. (a) The influenza (Flu)2C5 CD4 + T cell clone was stained with phycoerythrin (PE)-conjugated (right) or dextramer (left) reagents constructed with human leucocyte antigen (HLA)-DR1 monomers bearing the cognate influenza A virus HA 307–319 peptide (PKYVKQNTLKLAT; red line) or the HIV-1 p24 Gag 299–312 peptide (DRFYKTLRAEQASQ; green line) as an irrelevant control. Mean fluorescence intensity (MFI) values are shown inset for each tetramer/dextramer reagent and unstained cells (black line). (b) Human peripheral blood mononuclear cells (PBMCs) (10 6 ) were spiked with clonal Flu2C5 cells (10 4 ) and stained with the indicated tetramers (right) or dextramers (left), the viability dye Aqua and monoclonal antibodies against the surface markers CD3, CD4, CD8, CD14 and CD19. Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells prior to display as CD4 versus tetramer/dextramer. MFI values are shown inset.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Immunopeptidomics, Staining, Construct, Virus, Control, Fluorescence, Bioprocessing

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Dextramers bind a type I diabetes-derived CD8 + T cell clone substantially better than tetramers and also distinguish such cells from other T cells in a spiked peripheral blood mononuclear cell (PBMC) setting. (a) The 3F2 CD8 + T cell clone was stained with phycoerythrin (PE)-conjugated tetramer (right) or dextramer (left) reagents constructed with human leucocyte antigen (HLA)-A2 monomers bearing the cognate preproinsulin (PPI) 15–24 peptide (ALWGPDPAAA) or the melanoma antigen (MAGE) A3 168–176 peptide (YLEYRQVPV) as an irrelevant control. Different amounts of tetramer or dextramer were used as indicated with respect to the monomeric peptide-major histocompatibility complex (pMHC) component. Mean fluorescence intensity (MFI) values are shown for each condition. (b) Human PBMCs (10 6 ) were spiked with clonal 3F2 cells (10 4 ) and stained with the indicated tetramers (right) or dextramers (left), the viability dye Aqua and monoclonal antibodies against the surface markers CD3, CD4, CD8, CD14 and CD19. Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells prior to display as CD8 versus tetramer/dextramer. Clonal 3F2 cells were easily distinguishable on the basis of brighter CD8 expression. Reagent quantities with respect to the pMHC-I component (black) and MFI values (red) are shown inset.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Derivative Assay, Staining, Construct, Control, Immunopeptidomics, Fluorescence, Bioprocessing, Expressing

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Superior ex vivo detection of antigen-specific CD8 + T cells with dextramers. Human leucocyte antigen (HLA)-A2 + peripheral blood mononuclear cells (PBMCs) from non-type I diabetes (T1D) donors (left, middle) or T1D patients (right) were stained with tetramer or dextramer reagents constructed with HLA-A2 monomers bearing the Epstein–Barr virus (EBV) BMLF-1 259–267 peptide (GLCTLVAML; left), the MART-1 26–35 heteroclitic peptide (ELAGIGILTV; middle) or the preproinsulin (PPI) 15–24 peptide (ALWGPDPAAA; right). Each symbol and line combination denotes a different donor. Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells prior to analysis as CD8 versus tetramer/dextramer. Detected antigen-specific CD8 + T cell frequencies (top) and mean fluorescence intensity (MFI) values (bottom) are shown. Representative flow cytometry plots are shown in Supporting Information . The difference in MFI and percentage of CD8 + T cells detected between dextramers and tetramers is significantly different ( P ≤ 0·01: paired, two-tailed t -test).

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Ex Vivo, Staining, Construct, Virus, Fluorescence, Flow Cytometry, Two Tailed Test

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Peptide–major histocompatibility complex (pMHC) dextramer staining is enhanced by protein kinase inhibitor (PKI) treatment. The ILA1 CD8 + T cell clone was stained with phycoerythrin (PE)-conjugated tetramer (right) or dextramer (left) reagents constructed with human leucocyte antigen (HLA)-A2 + monomers bearing the cognate human telomerase reverse transcriptase (hTERT) 540–548 peptide (ILAKFLHWL), variants thereof with defined T cell receptor (TCR)-pMHC-I affinities as indicated in the key, or the HIV-1 Pol 476–484 peptide (ILKEPVHGV) as an irrelevant control after pretreatment with (bottom) or without (top) 50 nM dasatinib. Mean fluorescence intensity (MFI) values are shown inset.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Immunopeptidomics, Staining, Construct, Reverse Transcription, Control, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Superior recovery of antigen-specific CD8 + T cells with peptide–major histocompatibility complex (pMHC) dextramer + protein kinase inhibitor (PKI). Human peripheral blood mononuclear cells (PBMCs) (10 6 ) were spiked with clonal ILA1 cells (10 4 ), pretreated with or without 50 nM dasatinib (+/− PKI) and then stained with phycoerythrin (PE)-conjugated tetramer (lower) or dextramer (upper) reagents as indicated (details in Fig. ), followed by the viability dye Aqua and monoclonal antibodies against the surface markers CD3, CD4, CD8, CD14 and CD19. Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells prior to display as CD8 versus tetramer/dextramer. In each column, the red box is positioned on the antigen-specific CD8 + T cell population that stains most brightly to aid visual comparison.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Immunopeptidomics, Staining, Bioprocessing, Comparison

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Improved staining of autoimmune T cell clones with peptide–major histocompatibility complex (pMHC) dextramer and protein kinase inhibitor (PKI). The 4C6 and 1E6 CD8 + T cell clones were stained with phycoerythrin (PE)-conjugated tetramer or dextramer reagents constructed with human leucocyte antigen (HLA)-A24 monomers bearing the LWMRLLPLL peptide or HLA-A2 monomers bearing the ALWGPDPAAA peptide, respectively, after pretreatment with or without 50 nM dasatinib (+/− PKI). Irrelevant allotype-matched control reagents were constructed with HLA-A24–AYAQKIFKIL and HLA-A2–NLVPMVATV monomers. FMO = fluorescence minus one.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Staining, Clone Assay, Immunopeptidomics, Construct, Control, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Superior ex vivo detection of antigen-specific CD8 + T cells with a combinatorial staining approach using a protein kinase inhibitor (PKI) and peptide–major histocompatibility complex (pMHC) dextramer. Human leucocyte antigen (HLA)-A2 + peripheral blood mononuclear cells (PBMCs) from healthy controls were stained with phycoerythrin (PE)-conjugated tetramer (right) or dextramer (left) reagents constructed with HLA-A2 monomers bearing the MART-1 26–35 heteroclitic peptide (ELAGIGILTV) after pretreatment with or without 50 nM dasatinib (+/− PKI). Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells prior to display as CD8 versus tetramer/dextramer. Detected antigen-specific CD8 + T cell frequencies (black) and mean fluorescence intensity (MFI) values (red) are shown inset.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Ex Vivo, Staining, Immunopeptidomics, Construct, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Peptide–major histocompatibility complex (pMHC) dextramers are compatible with high-resolution flow cytometry. Human leucocyte antigen (HLA)-A2 + peripheral blood mononuclear cells (PBMCs) from healthy controls were stained with phycoerythrin (PE)-conjugated or dextramer reagents constructed with HLA-A2 monomers bearing the CMV pp65 495–503 peptide (NLVPMVATV), followed by the viability dye ViViD and monoclonal antibodies against the surface markers CD3, CD8, CD14, CD19, CD27, CD45RO, CD57, CD69 and CCR7. Gates were set serially on lymphocytes, single cells and live CD3 + CD14 − CD19 − cells (a) prior to display as CD8 versus tetramer/dextramer (b,c). Boolean gating was carried out to exclude artefacts and fluorochrome aggregates as indicated (a). Yellow or green dots superimposed on density plots showing the phenotypical profile of the overall CD8 + T cell population depict individual antigen-specific CD8 + T cells stained with dextramer or tetramer, respectively (d–i). The following bivariate analyses are shown: CD27 versus CD45RO (d,e); CD57 versus CCR7 (f,g); and CD69 versus CCR7 (h,i).

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Immunopeptidomics, Flow Cytometry, Staining, Construct, Bioprocessing

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Dextramers can be used to detect activated T cells during an intracellular cytokine assay. A 10-day-old enriched T cell line specific for the influenza epitope, GILGFVFTL (matrix protein residues 58–66), was cultured with or without cognate peptide for 5 h in the presence of monensin and brefeldin A. Cells were subsequently stained with cognate [human leucocyte antigen (HLA)-A2 GILGFVFTL] or irrelevant (HLA-A2–ALWGPDPAAA) dextramer or tetramer, cell surface markers (CD8, CD3, CD19, CD14), a viability stain and intracellularly for interferon (IFN)-γ. Unstimulated cells were stained with cognate dextramer (a) or tetramer (b), with the gate shown based on staining with an irrelevant dextramer or tetramer. The percentage of staining of IFN-γ + cells (c: red gate, based on a fluorescence minus 1) with cognate dextramer (red) or tetramer (green) is based upon the staining of the same IFN-γ + cells with an irrelevant dextramer (blue) or tetramer (orange) (d). The percentage of gated cells shown inset (a–c).

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Cytokine Assay, Cell Culture, Staining, Fluorescence

Journal: Clinical and Experimental Immunology

Article Title: Comparison of peptide–major histocompatibility complex tetramers and dextramers for the identification of antigen-specific T cells

doi: 10.1111/cei.12339

Figure Lengend Snippet: Similar visual staining patterns with peptide–major histocompatibility complex (pMHC) dextramers and tetramers. The NLV2F3 CD8 + T cell clone was stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue), followed by Alexa488-conjugated tetramer or fluorescein isothiocyanate (FITC)-conjugated dextramer reagents constructed with human leucocyte antigen (HLA)-A2 monomers bearing the cytomeglovirus (CMV) pp65 495–503 peptide (NLVPMVATV). Staining conditions are indicated; dasatinib was used at a concentration of 50 nM. Cells were visualized using a Leica DM LB2 fluorescent microscope.

Article Snippet: Cells were prepared for intracellular cytokine staining by incubation with Cytofix/CytopermTM (BD Biosciences) according to the manufacturer's instructions (including wash steps), followed by 20 min on ice with mouse αhuman interferon (IFN)-γ antibody (45-15; Miltenyi Biotec).

Techniques: Staining, Immunopeptidomics, Construct, Concentration Assay, Microscopy

Journal:

Article Title: Age-Related Changes in the Osteogenic Differentiation Potential of Mouse Bone Marrow Stromal Cells

doi: 10.1359/JBMR.080304

Figure Lengend Snippet: Characterization of BMSCs. (A) Marrow cell cultures (top) and antibody-enriched BMSCs from 18-mo-old mice (bottom) were immuno-labeled with FITC-conjugated monoclonal antibodies as indicated and detected using a fluorescence microscope. (B) FACS analysis of enriched BMSCs. BMSCs from indicated ages of mice were labeled with FITC-conjugated monoclonal antibodies (CD45, CD11b, and Sca-1, respectively) and analyzed. Percentages of cells positive for CD45, CD11b, and Sca-1 are shown. This experiment was performed in triplicate. (C) Multipotentiality of enriched BMSCs. BMSCs isolated from 18-mo-old mice were exposed to osteogenic, adipogenic, and myogenic differentiation media. (a) Cells were treated with osteogenic supplements for 21 days and stained with ARS for mineralized bone nodules. (b) Cells were treated with adipogenic induction media for 2 days, cultured in maintenance media for 14 days, and stained with Oil Red O. (c) Cells were treated with 5-azacytidine (5-Aza) for 24 h, cultured in regular growth media for 21 days, and immuno-labeled with indicated antibodies. These experiments were performed a minimum of three times in triplicate. (d) Bone tissue labeled with GFP antibody to show osteoblast-like lining cells (arrows) 6 wk after injection of GFP-BMSC into mouse tibias. Star indicates GFP-BMSC embedded in the bone. These experiments were repeated at least three times with similar results.

Article Snippet: Magnetic beads conjugated with anti-mouse CD11b (#558013) and CD45R/B220 (#551513) monoclonal antibodies and CD11b- (#557396) and CD45-FITC (#553079) monoclonal antibodies were purchased from BD Biosciences Pharmingen; Pan DC (#130-092-465) and stem cell antigen-1 (Sca-1) microbeads (130–092–529) were from Miltenyi Biotec.

Techniques: Labeling, Bioprocessing, Fluorescence, Microscopy, Isolation, Staining, Cell Culture, Injection

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: CD133 expression in the human gallbladder cancer cell line GBC-SD following cell sorting using immunomagnetic beads. (A) Transmembrane CD133 expression in CD133 + and CD133 − groups. CD133 was labeled with red fluorescence as indicated by arrows (magnification, ×200). (B) Proportion of the CD133 + subset in the CD133 + and CD133 − groups in GBC-SD cells, determined using flow cytometry. (C) Detection of CD133 mRNA expression using polymerase chain reaction. GADPH was used as the control. (D) Detection of CD133 protein expression using western blot analysis. GADPH was used as the loading control. CD, cluster of differentiation.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing, FACS, Labeling, Fluorescence, Flow Cytometry, Polymerase Chain Reaction, Control, Western Blot

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Colony formation assay and in vivo tumor formation assay. (A) Shape of the colony formed by the CD133 + subset (magnification, ×10). (B) Colony formation efficiency in CD133 + and CD133 − groups. (C) Representative images of the xenograft tumors formed by CD133 + cells, with representative HE staining and CD133 IHC staining, as observed under an inverted microscope (magnification, ×200). The arrow indicates CD133 + cells. CD, cluster of differentiation; H&E, hematoxylin and eosin; IHC, immunohistochemistry.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Colony Assay, In Vivo, Tube Formation Assay, Staining, Immunohistochemistry, Inverted Microscopy

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Cell biology characteristics of cells in the CD133 + and CD133 − groups. (A) Comparison of the proliferative ability between the CD133 + and CD133 − groups. (B) Inhibiting rate of cell growth in the CD133 + and CD133 − groups 72 h after treatment with 5-fluorouracil or gemcitabine. CD, cluster of differentiation.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Comparison

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Cell invasion and EMT. (A) Number of transmembrane cells in the CD133 + and CD133 − groups, observed under an inverted microscope (magnification, ×200). (B) Quantitative analysis of the number of invasive cells. (C) Expression of EMT-associated proteins in the CD133 + and CD133 − groups. (D) Quantitative analysis of mRNA expression of EMT-associated genes in the CD133 + and CD133 − groups. EMT, epithelial-mesenchymal transition; CD, cluster of differentiation.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Inverted Microscopy, Expressing

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Expression of stem cell-associated genes. (A) DNA electrophoresis of stem cell-associated genes in the CD133 + and CD133 − groups, determined using polymerase chain reaction. (B) Quantitative analysis of mRNA expression level of stem cell-associated genes. GADPH was used as the loading control. CD, cluster of differentiation; CXCR4, C-X-C motif chemokine receptor 4; p-, phosphorylated; Akt, protein kinase B; Erk, extracellular signal-regulated kinase; SDF-1α, stromal cell-derived factor 1α.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Control, Derivative Assay

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: Regulation of the CXCR4/Akt/CD133 signaling pathway. (A) Differences in CXCR4/Akt/CD133 protein expression between the CD133 + and CD133 − group. (B) Protein expression of CXCR4/Akt/CD133 as induced by treatment with SDF-1α at different concentrations (25, 50, 100, and 200 ng/ml) and for different durations (15, 30 min, 1, 2, and 24 h).

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing

Journal: Oncology Letters

Article Title: Isolation and identification of tumor-initiating cell properties in human gallbladder cancer cell lines using the marker cluster of differentiation 133

doi: 10.3892/ol.2017.7159

Figure Lengend Snippet: (A) CD133 and CXCR4 mRNA expression profiles in the CD133 + and CD133 − groups following treatment with SDF-1α/AMD3100/LY294002. (B) CXCR4, p-Akt, Akt, p-Erk, Erk, and CD133 protein expression profiles in the CD133 + and CD133 − groups following treatment with SDF-1α/AMD3100/LY294002. The CXCR4/Akt/CD133 axis and its potential function. CXCR4, C-X-C motif chemokine receptor 4; Akt, protein kinase B; CD, cluster of differentiation; SDF-1α, stromal cell-derived factor 1α; p-, phosphorylated; Erk, extracellular signal-regulated kinase.

Article Snippet: Subsequently, the PVDF membrane was blocked with 5% non-fat milk powder diluted in TBST at room temperature for 2 h and incubated with the following primary monoclonal antibodies (all diluted to 1:200): Mouse anti-human CD133 (cat no. 130-105-226; Miltenyi Biotec GmbH), rabbit anti-human Snail (cat no. 3879s), rabbit anti-epithelial (E-)cadherin (cat no. 3195s), mouse anti-human neural (N-)cadherin (cat no. 13116s), rabbit anti-human Akt (cat no. 4685s), rabbit anti-human phosphorylated (p-)Akt (cat no. 4060s), rabbit anti-human extracellular signal-regulated kinase (Erk; cat no. 4695s), rabbit anti-human p-Erk (cat no. 4370s) (all from Cell Signaling Technology, Inc.), or rabbit anti-human CXCR4 (cat no. ab124824; Abcam, Cambridge, MA, USA) at 4°C overnight.

Techniques: Expressing, Derivative Assay

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet:

Article Snippet: Mouse anti-A2B5 supernatant, clone 105 , ATCC , Cat#CRL-1520; RRID: CVCL_7946.

Techniques: In Vivo, In Vitro

Journal: Cell reports

Article Title: Human Glial Progenitor Cells Effectively Remyelinate the Demyelinated Adult Brain

doi: 10.1016/j.celrep.2020.107658

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse anti-A2B5 supernatant, clone 105 , ATCC , Cat#CRL-1520; RRID: CVCL_7946.

Techniques: Marker, In Vitro, Recombinant, Clinical Proteomics, Software, Microscopy, Lysis

Journal: STAR Protocols

Article Title: Preparation of monovalent follistatin-like 3-Fc-fusion protein and evaluation of its effects on muscle mass in mice

doi: 10.1016/j.xpro.2021.100839

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-human IgG Fc , Bethyl Laboratories , Cat#A80-105A; RRID: AB_67482.

Techniques: Recombinant, Antibody Purification, Expressing, Enzyme-linked Immunosorbent Assay, Software, Cell Counting, Microscopy

Journal: bioRxiv

Article Title: A standardized method to purify cardiomyocytes from individual mouse hearts of any age

doi: 10.1101/2021.09.30.462562

Figure Lengend Snippet: Flow diagram outlining the isolation and purification procedure (<1 hr). For in situ fixation of cardiac cells, the heart is perfused with paraformaldehyde immediately after step 2. The cardiomyocyte enrichment (step 4) is omitted for neonatal cardiac cells. See Methods for further details.

Article Snippet: Neonatal cardiomyocytes were purified immediately after the isolation step also by immunomagnetic bead-based negative selection (using the Neonatal Cardiomyocyte Isolation Kit, Miltenyi Biotec).

Techniques: Isolation, Purification, In Situ

Journal: bioRxiv

Article Title: A standardized method to purify cardiomyocytes from individual mouse hearts of any age

doi: 10.1101/2021.09.30.462562

Figure Lengend Snippet: A, Representative immunofluorescence images of C57BL/6J P10 murine cardiac cells stained to identify nucleated (DNA, TO-PRO-3, blue) cardiomyocytes (cardiac troponin T, cTnT, green) and endothelial cells (isolectin B4, IB4, red) following cardiomyocyte isolation, enrichment and purification steps, including the CD31 Ab-bead bound fraction. Scale bar is 100μm. B, Quantification from immunocytochemistry of cardiac cell types after each step of isolation and purification from C57BL/6J P2 (n= 3, ~2000 cells/n), P10 (n=3-4, ~1500 cells/n), P13 (n=3, ~1500 cells/n), or adult (n=3, ~1500 cells/n) hearts (see representative images in Online – ). Cardiomyocytes, CMs (TO-PRO-3+, cTnT+); endothelial cells, ECs, (TO-PRO-3+, IB4+); other cells (TO-PRO-3+, cTnT-, IB4-). C, Representative flow cytometric data evaluating the percentage of cells (propidium iodide, PI+) that were cardiomyocytes (cTnT+) compared to non-myocytes (cTnT-) from P2, P10 and P70 PFA-fixed cardiac cell populations after the isolation and purification steps. Approximately 100,000-500,000 cells were analysed per sample and percentages were calculated as a total of the nucleated cell population (PI+). The gating strategy was determined per sample based on their corresponding unstained and single stained fractions (PI+ only, cTnT+ only).

Article Snippet: Neonatal cardiomyocytes were purified immediately after the isolation step also by immunomagnetic bead-based negative selection (using the Neonatal Cardiomyocyte Isolation Kit, Miltenyi Biotec).

Techniques: Immunofluorescence, Staining, Isolation, Purification, Immunocytochemistry

Journal: bioRxiv

Article Title: A standardized method to purify cardiomyocytes from individual mouse hearts of any age

doi: 10.1101/2021.09.30.462562

Figure Lengend Snippet: A, qRT-PCR gene expression of cardiac cell markers after cardiomyocyte isolation, enrichment and purification steps, collected from: neonatal P2 (n=3); infant P10 (n=3); and adult P70 hearts (n=2-5). Genes were normalized to Gapdh and fold-change calculated relative to the isolation fraction (2 −ΔΔCt method), shown as % (mean ± SEM). Cardiomyocyte markers (orange): Myh6 , α-myosin heavy chain and Tnnt2 , cardiac Troponin T; Endothelial cell markers (purple): Pecam1 , Platelet And Endothelial Cell Adhesion Molecule 1, and Vwf , von Willebrand Factor; Fibroblast markers (blue): Ddr2 , Discoidin Domain Receptor 2 and Col1a1 , Collagen Type I Alpha 1 Chain. B, Representative brightfield images of freshly-purified high-quality cardiomyocytes (~80-85% rod-shaped CMs) from C57BL/6J neonatal (P2), infant (P10), and adult (8-10-week-old) mouse hearts. Cells were dispersed onto slides and images were taken at 5X, 20X, 40X objectives on a Leica microscope and 4X on a Nikon Eclipse TS100 microscope.

Article Snippet: Neonatal cardiomyocytes were purified immediately after the isolation step also by immunomagnetic bead-based negative selection (using the Neonatal Cardiomyocyte Isolation Kit, Miltenyi Biotec).

Techniques: Quantitative RT-PCR, Expressing, Isolation, Purification, Microscopy

Journal: bioRxiv

Article Title: A standardized method to purify cardiomyocytes from individual mouse hearts of any age

doi: 10.1101/2021.09.30.462562

Figure Lengend Snippet: A, The percentage of rod-shaped vs round-shaped C57BL/6J murine ventricular cardiomyocytes fixed in situ or post-isolation were counted from neonatal (P2), infant (P10 and P13), and adult (P70) preparations (n=2-4, 120-420 cardiomyocytes) presented as mean ± SEM. B, Cardiomyocyte area and number of nuclei per cell (see ) were evaluated from confocal immunofluorescence images of in situ fixed cardiac cells at postnatal ages: P2, P10, P13 and P70 (n= 2-3, ~110-300 cardiomyocytes per age). ImageJ was used to calculate cardiomyocyte area (μm 2 ) by planimetry. C, Representative images of in situ fixed cardiomyocytes are at postnatal ages P2, P10, P13, and P70. Cells were immunostained with the cardiomyocyte-specific marker, cTnT (green), and nuclei with DNA dye, TO-PRO-3 (blue). Scale bar is 100 μm. D, Neonatal (P2) cardiomyocytes fixed in situ were captured undergoing cytokinesis as shown by positive immunostaining of the midbody with Aurora B Kinase antibody (red). Scale bar is 20 μm. C and D, Representative cells were selected and grouped in a horizontal position. E, Heart weight (HW, n=6-8, mean ± SD, closed circles) and cardiomyocyte area (n=2-3, open circles) were compared along the trajectory of postnatal cardiac growth: P2, P10, P13 and P70 (adult male) ages.

Article Snippet: Neonatal cardiomyocytes were purified immediately after the isolation step also by immunomagnetic bead-based negative selection (using the Neonatal Cardiomyocyte Isolation Kit, Miltenyi Biotec).

Techniques: In Situ, Isolation, Immunofluorescence, Marker, Immunostaining

Journal: bioRxiv

Article Title: A standardized method to purify cardiomyocytes from individual mouse hearts of any age

doi: 10.1101/2021.09.30.462562

Figure Lengend Snippet: A, Schematic of the experimental design. Cardiomyocytes were purified from one postnatal day 2 male and female heart per litter of 6-8 C57BL/6J mouse pups (x4 litters, L1-L4). Poly(A) RNA was extracted from cardiomyocytes and sequenced using the NovaSeq 6000 Sequencing platform generating ~22 M reads per heart. Created with BioRender.com. B, Body weights (BW) were similar between male (?) and female (o) littermates (n=4 litters). C, Hierarchical clustering dendrogram (Ward method) based on the expression of 12,854 genes (>1 count per million, cpm, in four samples) shows clustering by litters but not sex. D, Heatmap shows nine differentially expressed genes (DEGs, FDR<0.005) between male and female cardiomyocytes, five genes were on chromosome Y (ChrY) and four on chromosome X (ChrX). E, Abundance (cpm) of the nine DEGs shown as dot plot and mean, ordered from highest (left) to lowest (right) counts in male (□) and female (○) cardiomyocytes.

Article Snippet: Neonatal cardiomyocytes were purified immediately after the isolation step also by immunomagnetic bead-based negative selection (using the Neonatal Cardiomyocyte Isolation Kit, Miltenyi Biotec).

Techniques: Purification, Sequencing, Expressing