Journal: Neuro-Oncology

Article Title: Dissecting inherent intratumor heterogeneity in patient-derived glioblastoma culture models

doi: 10.1093/neuonc/now253

Figure Lengend Snippet: Floating cells display enhanced mesenchymal properties. (A) MGG6 NS and FC mRNA were analyzed by qRT-PCR for different mesenchymal markers including CD44, vimentin, YKL-40, N-cad, and MMP2. (B) Cell lysates from different cultures were analyzed by western blotting for pSTAT3, STAT3, CD44, C/EBPβ, and β-actin. (C) FACS analysis showing proportion of CD44 positive cells in FC and their corresponding NS from different GSC cultures. (D) MGG29 NS were fractionated into CD44high and CD44low subpopulation by FACS and predominant cells were established and labeled with GFP and mCherry, respectively. Left: fluorescence analysis of a mixture of these 2 subpopulations cultured in serum; middle: FC collected and analyzed for both GFP and mCherry; right: flow cytometry of CD44high cells in both GFP and mCherry populations. (E) NS and FC were differentiated using StemPro Chondrogenesis Differentiation Kit and stained with fast green and safranin O (positive, #; negative, +). (F) NS and FC were cultured as a monolayer and their ability to invade/migrate was evaluated using scratch healing assay. Bright field micrographs are showing for different groups. Scale bar, 50 μm.

Article Snippet: CD44 expression was measured in 500000 cells dissociated with 2 mM EDTA and incubated in 80 μL PBS/0.1% bovine serum albumin at 4°C for 30 min in the dark with mouse monoclonal anti-human CD44 antibody conjugated to fluorescein isothiocyanate (FITC, 1:10; Miltenyi Biotec) or respective mouse immunoglobulin G1 control in the presence of FcR blocking reagent (1:5).

Techniques: Quantitative RT-PCR, Western Blot, Labeling, Fluorescence, Cell Culture, Flow Cytometry, Staining

Journal: Cancers

Article Title: Ex Vivo Behaviour of Human Bone Tumor Endothelial Cells

doi: 10.3390/cancers5020404

Figure Lengend Snippet: Characterization of TECs. ( A ) Expression of endothelial markers in a representative TEC line and HAEC cells, used as a control detected by FACS analysis. FACS analysis of HAECs, and TEC 1, with CD31 antibody at 1st passage and 3th passage. Expression of CD44 antigen in HAECs and TEC 1. ( B ) Bar graph expressing percentage of positive cells to specific antigens analyzed by FACS. Data are expressed as mean ± SE of 10 different TEC lines grouped by histology and three control cell lines. Each experiment was repeated three times. TECs vs . HAECs ( p < 0.001). IgG was used as negative control. ( C ) Representative example of TECs analyzed with CD146 antibody.

Article Snippet: For cytofluorimetric analysis the primary antibodies used were: anti CD31-FITC (R&D Systems, Inc. Minneapolis, MN, USA), anti VE-cadherin-PE (Santa Cruz Biotechnology, Inc. Milan, Italy), anti CD133-PE (Miltenyi Biotech), anti fusin-PE (Santa Cruz Biotechnology), anti CD14-PE (R&D Systems), anti CD44-FITC (R&D Systems), anti CD13-PE/Cy5 (Chemicon International, Temecula, CA, USA), anti CD34-PE, anti CD105-PE, anti CD45-PE, anti CD90-PE, anti CD131-PE, anti CCR7-PE, anti CD133-PE (Miltenyi Biotech.), anti CD146-PE (Miltenyi Biotech.) and anti CD309-PE (Miltenyi Biotech.).

Techniques: Expressing, Control, Negative Control