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Int. J. Mol. Sci. 2018, 19, 1107 10 of 17 significant either in cases of SCC-25 or Detroit 562 cells. As visible on Figure 4A,B) Detroit 562 cells have a two-time higher basic cell clone producing capacity than SCC-25 cells, but the MB + laser treatment reduces this capacity to 19 ± 26 colonies, which is lower than that of 38 ± 8 colonies of SCC-25 cells. Immunohistochemical stainings of agarose and paraffin embedded cell pellets of Detroit 562 and SCC-25 cells revealed a high representation of cancer stem cells in the Detroit 562 culture, which is highly consistent with the result that Detroit 562 cell culture has a high clone producing capacity in normal culture conditions. It is particularly interesting, that a short time high concentrated MB treatment followed by 8 min of light exposition, which are, at least from the point of view of time, clinically also endurable, delivered a highly efficient killing of tumorigenic cancer stem cells. 4. Materials and Methods 4.1. Cell Lines Detroit 562 cells were purchased from Cell Lines Service (CLS Cell Lines Service, Heidelberg, Germany) and were routinely cultured in DMEM/F12 (PAA) supplemented with 10% FBS (PAA), 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin at 37 ◦C and 5% CO2 [39]. SCC-25 cells were acquired from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany, DSMZ no.: ACC 617), and were cultured in DMEM/F12 medium, supplemented with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin [34,63–66]. 4.2. Evaluation of Cancer Stem Cells—Related Markers in Cultured Cell Lines Routinely cultured cell lines (2–4 × 106) were collected by centrifugation and embedded as cell pellet in agarose as published before [67], modified as follows: Cells were harvested by centrifugation at 290 g for 10 min at 4 ◦C, and the resulting pellet was fixed in 10 mL neutral buffered 4% formaldehyde solution (SAV liquid production, Flintsbach am Inn, Germany). After fixation the cells were centrifuged by 400 g for 10 min at room temperature. The cell pellet was resuspended in 300 μL PBS, transferred to Eppendorf tube (1.5 mL), and kept on ice. Low melting point agarose (with gelling temperature point 34–37 ◦C) was prepared in PBS as 3% solution in labor glassware by microwave warming and it was equilibrated in a thermoblock to 65 ◦C for at least 30 min. The 300 μL PBS—cell suspension was also equilibrated to 65 ◦C for not more than 10 min. 600 μL melted equilibrated agarose was pipetted to the cell suspension, followed by spinning at 2000× g for 5 min at room temperature. After that, the tube was placed on ice, the cell pellet was trimmed and it was placed in embedding cassette. The cell pellet in the cassette was stored in PBS containing 0.05–0.1% sodium azide until embedded in paraffin, which was done using a Histos 5 (Histocom, Wiener Neudorf, Austria) paraffin embedding system, following the instructions of the manufacturer. After embedding, cell pellets were serially sectioned at 5 μm thickness using a HM 355S microtome (Microm, Walldorf, Germany), affixed onto SuperfrostTM Plus slides (Menzel, Braunschweig, Germany), and used for immunohistochemistry. Immunohistochemistry was performed utilizing a Ventana Roche® Discovery Immunostainer (Roche, Mannheim, Germany), applying a manufacturer supplied FISH procedure. Antigen retrieval was performed by epitope unmasking via a heat induction methodology performed while the sections were immersed in EDTA buffer (Cell Conditioning Solution CC1, Ventana 950–124; (Roche, Mannheim, Germany). Cell pellet affixed slides were incubated with appropriate primary antibodies (CD44, mouse monoclonal (Antibodies Online, Aachen, Germany, Cat. Nr. ABIN1020059), and Nanog rabbit monoclonal (Cell Signaling Technology; Leiden, The Netherlands, Cat. Nr. 4903)) at 37 ◦C for 1 h. The primary antibodies were detected with anti-mouse or anti-rabbit Alexa 488 or Alexa 594 conjugated secondary antibodies (Invitrogen, Eugene, OR, USA) incubating for 30 min in the Ventana Discovery immunostainer. In addition mouse monoclonal p75NTR antibody (Sigma, Vienna, Austria, Cat. Nr. N5408) was also used as a single staining detected by Alexa 488 conjugated secondary anti–mouse antibody. The Alexa fluorochrome conjugated secondary antibody signals were detectedPDF Image | Photodynamic Low Level Laser Squamous Cell Carcinoma
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