Journal: Bio-protocol

Article Title: Human Schwann Cells in vitro III. Analytical Methods and a Practical Approach for Quality Control

doi: 10.21769/BioProtoc.4840

Figure Lengend Snippet: Useful antibodies to characterize human Schwann cells (hSCs) and non-glial cells established in cell culture. These antibodies were validated using traditional adult nerve-derived hSC monocultures. Staining with NGFR, Sox10, and S100B antibodies, alone or together with fibronectin, SMA (α-smooth muscle actin), and FAP (fibroblast activation protein/seprase) antibodies, is recommended for the initial characterization of hSCs and non-glial cells. We have not found a ubiquitous non-glial cell marker. Protocol 1C is suitable for staining with all antibodies except for anti-O1 and anti-O4, which can be accomplished only in live cells (Protocol 1B). A 1:200–1:500 starting dilution is suitable for most antibodies; the optimal concentration should be determined by the end user in the target cells. (*) Indicates that the antibodies are produced from hybridoma cell lines. MBP: myelin basic protein; MAG: myelin-associated glycoprotein; MPZ: myelin protein zero; GFAP: glial fibrillary acidic protein.

Article Snippet: FAP Cell membrane , Rabbit Monoclonal. Cell Signaling, catalog number: 66562. , Expressed at high levels in a proportion of non-glial cells. hSCs do not typically express FAP..

Techniques: Cell Culture, Staining, Activation Assay, Marker, Concentration Assay, Produced, Membrane, Isolation, Immunostaining

Journal: Bio-protocol

Article Title: Human Schwann Cells in vitro III. Analytical Methods and a Practical Approach for Quality Control

doi: 10.21769/BioProtoc.4840

Figure Lengend Snippet: Quick staining protocols for human Schwann cell (hSC) validation in culture. Table 2 presents simple strategies for verifying both the human and the SC background of cultured cells. Different antibody combinations using human-selective and multispecies-selective NGFR antibodies in combination with other stains are proposed. NGFR-8737 (mouse monoclonal), S100B (rabbit polyclonal), and GFAP (rabbit polyclonal) antibodies are described in Table 1 . HNA monoclonal antibodies are described in Materials and Reagents.

Article Snippet: FAP Cell membrane , Rabbit Monoclonal. Cell Signaling, catalog number: 66562. , Expressed at high levels in a proportion of non-glial cells. hSCs do not typically express FAP..

Techniques: Staining, Biomarker Discovery, Cell Culture, Bioprocessing, Derivative Assay

Journal: Journal of cell science

Article Title: Stromal fibroblasts from the interface zone of human breast carcinomas induce an epithelial-mesenchymal transition-like state in breast cancer cells in vitro.

doi: 10.1242/jcs.072900

Figure Lengend Snippet: Fig. 2. Comparison of primary fibroblasts from each breast cancer tissue zone based on the new three-zone concept of tumor margins. (A)Normal zone fibroblasts (NFs), interface zone fibroblasts (INFs), cancer-associated fibroblasts (CAFs) and MCF7 cells in tissue sections were examined by phase-contrast microscopy and immunostaining for vimentin, cytokeratin 5 or a wide-spectrum cytokeratin; nuclei were stained with DAPI. The fibroblasts from each zone had an identical, long, spindle-shaped morphology and exhibited vimentin expression, whereas they were negative for cytokeratin 5 and cytokeatin. By contrast, MCF7 cells were negative for vimentin and positive for cytokeratin 5 and cytokeatin. (B)NFs, INFs and CAFs seeded at the same density (2000 cells/well) in 96-well plates were assayed for cell viability after 5 days in culture using Cell Counting Kit-8. Cell numbers were calculated by reference to a standard curve obtained under the same experimental conditions. Fibroblasts from the interface zone exhibited significantly greater growth rates compared with NFs and CAFs (P<0.01). The data were derived from three independent experiments. (C)NFs, INFs and CAFs grown to confluence were routinely collected and apoptosis was measured by Annexin/PI staining and flow cytometry analysis. The percentages of apoptotic cells were graphed and the data were derived from three independent experiments. A similar apoptosis rate was observed in each type of fibroblasts. (D)Fibroblast activation protein (FAP) expression was investigated by flow cytometry. A representative histogram of analysis and mean fluorescence intensity of FAP-positive cells in each type of fibroblast at 2, 5 or 10 population doublings (PDs) is shown. FAP was significantly overexpressed in INFs compared with NFs and CAFs at each PD (P<0.01); furthermore, for each fibroblast type, FAP was stably expressed at each PD (*P>0.05 versus 5 PDs and 10 PDs, #P>0.05 versus 10 PDs). The data were derived from three independent experiments. (E)Breast carcinoma sections were immunostained with anti-FAP antibody. FAP was stained specifically in fibroblasts, but this was only significant in the interface zone. Spindle cells are fibroblasts and the brown color indicates positive staining.

Article Snippet: Next, an anti-fibroblast activation protein (FAP) primary antibody (clone F11-24, Santa Cruz Biotechnology, Santa Cruz, CA) was added (1 g per 106 cells) and incubated for 30 minutes on ice in the dark.

Techniques: Comparison, Microscopy, Immunostaining, Staining, Expressing, Cell Counting, Derivative Assay, Flow Cytometry, Activation Assay, Fluorescence, Stable Transfection