Journal: Biomarkers in Cancer

Article Title: A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis

doi: 10.4137/BIC.S33542

Figure Lengend Snippet: List of genes localized close to the IP in sSOM.

Article Snippet: The fixed samples were incubated with blocking solution (PBS containing 10% FBS, 1% bovine serum albumin [BSA], and 0.02% NaN 3 ), followed by incubation with anti-CD44 mouse monoclonal antibody (Cell signaling Technology, diluted 1:100 in PBS containing 1% BSA) at 4°C overnight or with anti-caveolin 1 rabbit polyclonal antibody (Cell Signaling Technology, diluted 1:200) at room temperature for one hour.

Techniques: Membrane, Marker

Journal: Biomarkers in Cancer

Article Title: A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis

doi: 10.4137/BIC.S33542

Figure Lengend Snippet: ( A ) Immunostaining of glioma cells with anti-CD44 antibody. Nine glioma cell lines were stained with anti-CD44 antibody followed by the secondary antibody labeled with fluorescein isothiocyanate (FITC). The secondary antibody did not show any background staining. The observation was performed by confocal microscopy at a magnification of ×64. ( B ) Immunostaining of glioma cells with anti-caveolin-1 antibody. Nine glioma cell lines were stained with anti-caveolin-1 antibody followed by the secondary antibody labeled with FITC. The secondary antibody did not show any background staining. The observation was performed by confocal microscopy at a magnification of ×64. The experiment was repeated three times and the same results were obtained.

Article Snippet: The fixed samples were incubated with blocking solution (PBS containing 10% FBS, 1% bovine serum albumin [BSA], and 0.02% NaN 3 ), followed by incubation with anti-CD44 mouse monoclonal antibody (Cell signaling Technology, diluted 1:100 in PBS containing 1% BSA) at 4°C overnight or with anti-caveolin 1 rabbit polyclonal antibody (Cell Signaling Technology, diluted 1:200) at room temperature for one hour.

Techniques: Immunostaining, Staining, Labeling, Confocal Microscopy

Journal: Translational Vision Science & Technology

Article Title: Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

doi: 10.1167/tvst.11.2.33

Figure Lengend Snippet: Human corneal endothelial cells cultured at [O 2 ] 2.5 demonstrate similar growth characteristics to cells grown at [O 2 ] A . (A) Phase-contrast microscopy of HCEnC cultures shows similar cell morphology under both culture conditions. (B) Immunofluorescence imaging for zonula occludens (ZO-1, green ) with DAPI-stained nuclei ( blue ) demonstrates similar integrity of tight junctions under both conditions (representative images from HCEnC cultures from n = 4 corneas). Quantification of cell characteristics for the data from four corneas. ECD, coefficient of variation (COV), and hexagonality (6A). (C) Western blot comparing CD44 expression in HCEnCs from three different donors grown at the different oxygen concentrations. CD44 expression levels were normalized to α-tubulin expression, showing the variability between donor corneas. (D) Senescence-associated β-galactosidase staining ( black ) with DAPI-stained nuclei ( blue) shows similarities in percentage of senescent cells (mean ± SD: [O 2 ] A 58.3% ± 15.8%, and [O 2 ] 2.5 53.8% ± 21.4%).

Article Snippet: Membranes were blocked (10% goat serum for CD44 blot or 5% nonfat milk [Blotting-Grade Blocker, Bio-Rad] for HIF-1α blot) for one hour followed by incubation overnight at 4°C in primary antibodies (CD44 [E7K2Y] XP Rabbit monoclonal antibody, diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA; HIF-1α [D1S7W] XP Rabbit monoclonal antibody diluted 1:1000, Cell Signaling Technology).

Techniques: Cell Culture, Microscopy, Immunofluorescence, Imaging, Staining, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: CD44-initiated Cell Spreading Induces Pyk2 Phosphorylation, Is Mediated by Src Family Kinases, and Is Negatively Regulated by CD45

doi: 10.1074/jbc.m100158200

Figure Lengend Snippet: FIG. 2. Immobilized anti-CD44 antibody triggers tyrosine phosphorylation in CD452 BW cells. A, anti-phosphotyrosine West- ern blot (PY) of CD451 and CD452 BW cell lysates (CD451 and CD452, respectively) prepared from 105 cells immobilized on anti-CD44 anti- body, KM201 (CD44 mAb)-coated tissue culture plates after the indi- cated time points. B, anti-phosphotyrosine Western blot of CD452 BW cells added to BSA-coated wells (CD452/BSA) or to anti-CD44 coated wells with pretreatment of 10 mM PP2 for 30 min (CD452/CD44 mAb/ PP2). Anti-actin Western blot after stripping the same membranes is shown below each blot to indicate loading amounts (see “Experimental Procedures”). Molecular mass is indicated on the left in kDa.

Article Snippet: Antibody Immobilization—50 ml of purified anti-CD44 antibody KM201 (40 mg/ml) or 2% w/v bovine serum albumin (BSA, Life Technologies, Inc.) in phosphate-buffered saline (PBS), were coated on 96- well tissue culture plates at 37 °C for 3 h. Plates were washed 23 with PBS then blocked with 2% w/v BSA in PBS for another 2 h at 37 °C and washed 33 with PBS prior to use.

Techniques: Phospho-proteomics, Western Blot, Stripping Membranes

Journal: Translational Vision Science & Technology

Article Title: Effect of Physiological Oxygen on Primary Human Corneal Endothelial Cell Cultures

doi: 10.1167/tvst.11.2.33

Figure Lengend Snippet: Human corneal endothelial cells cultured at [O 2 ] 2.5 demonstrate similar growth characteristics to cells grown at [O 2 ] A . (A) Phase-contrast microscopy of HCEnC cultures shows similar cell morphology under both culture conditions. (B) Immunofluorescence imaging for zonula occludens (ZO-1, green ) with DAPI-stained nuclei ( blue ) demonstrates similar integrity of tight junctions under both conditions (representative images from HCEnC cultures from n = 4 corneas). Quantification of cell characteristics for the data from four corneas. ECD, coefficient of variation (COV), and hexagonality (6A). (C) Western blot comparing CD44 expression in HCEnCs from three different donors grown at the different oxygen concentrations. CD44 expression levels were normalized to α-tubulin expression, showing the variability between donor corneas. (D) Senescence-associated β-galactosidase staining ( black ) with DAPI-stained nuclei ( blue) shows similarities in percentage of senescent cells (mean ± SD: [O 2 ] A 58.3% ± 15.8%, and [O 2 ] 2.5 53.8% ± 21.4%).

Article Snippet: Membranes were blocked (10% goat serum for CD44 blot or 5% nonfat milk [Blotting-Grade Blocker, Bio-Rad] for HIF-1α blot) for one hour followed by incubation overnight at 4°C in primary antibodies (CD44 [E7K2Y] XP Rabbit monoclonal antibody, diluted 1:1000, Cell Signaling Technology, Danvers, MA, USA; HIF-1α [D1S7W] XP Rabbit monoclonal antibody diluted 1:1000, Cell Signaling Technology).

Techniques: Cell Culture, Microscopy, Immunofluorescence, Imaging, Staining, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/ijms23020877

Figure Lengend Snippet: ( A ) Representative immunohistochemical (IHC) staining of CD44 expression of responder and non-responder patients in the biopsy sections maintained in ex vivo culture. ( B ) Mean ± SD of CD44 labelling intensities, quantified using Metafer software (Metasystems, Altlussheim, Germany) for 15 responder and 13 non-responder patients. ( C ) Ratio of CD44 expression after 4 Gy irradiation compared with 0 Gy for responder and non-responder patients. Two slides and a minimum of 200 fields per slide were analysed per condition. Student’s t -test was used for statistical analyses ( ns p > 0.05, * p < 0.05).

Article Snippet: However, tissue sections were incubated with 1:100 CD44 rabbit monoclonal antibody (E7K2Y, Cell Signaling, Danvers, MA, USA) in EnVision FLEX antibody diluent overnight at 4 °C or with 1:70 pATM rabbit monoclonal antibody (phospho S1981, Abcam, Cambridge, UK) 2 h at 37 °C in TBS (Tris Buffered Saline), 1% BSA (Bovine Serum Albumin, Sigma (Merck, Darmstadt, Germany)).

Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Ex Vivo, Software, Irradiation

Journal: International Journal of Molecular Sciences

Article Title: CD44, γ-H2AX, and p-ATM Expressions in Short-Term Ex Vivo Culture of Tumour Slices Predict the Treatment Response in Patients with Oral Squamous Cell Carcinoma

doi: 10.3390/ijms23020877

Figure Lengend Snippet: Receiver Operating Characteristic (ROC) analyses for the different markers at basal levels (0 Gy) or after 4 Gy irradiation: 4 Gy/0 Gy ratio. Analyses were performed for all biomarkers individually or in combination.

Article Snippet: However, tissue sections were incubated with 1:100 CD44 rabbit monoclonal antibody (E7K2Y, Cell Signaling, Danvers, MA, USA) in EnVision FLEX antibody diluent overnight at 4 °C or with 1:70 pATM rabbit monoclonal antibody (phospho S1981, Abcam, Cambridge, UK) 2 h at 37 °C in TBS (Tris Buffered Saline), 1% BSA (Bovine Serum Albumin, Sigma (Merck, Darmstadt, Germany)).

Techniques: Irradiation