To determine fluorescence at the maximal migration rate, equal numbers of cells were directly seeded to wells of the lower chamber while only medium was added to the upper chamber

A p-benefit of <0.05 based on the ratio measurement and its associated error in addition to fold change cut of 2 (Cyc vs. Wt) was considered as significant. Expression data were submitted to GEO.Microarray data were validated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) using 3 independent biological samples of freshly isolated Sca-1 cells. In addition to selected genes (Bdnf, Ccl19, Ccl9, Crlf1, Cxcl13, Ptn, Sfrp2, Spp1, Wisp2), actin (Actb) was used as endogenous control. PCR amplification was performed using a 7900 HT Sequence Detection System (Applied Biosystems, CA, USA). Thermal cycling was as follows: 2 minutes at 50 10 minutes at 95, followed by 15 seconds at 95 and 1 minute at 60 for 45 cycles. Real-time PCR data were quantified using the SDS2.3 software package (Applied Biosystems) followed by relative quantification using the comparative Ct method (Ct method) [20] (S1 Table).Migration efficiency was determined by using a modified-Boyden chamber containing a nitrocellulose membrane of 8m pore size (Chemotaxis Assay, Millipore, Schwalbach, Germany). Freshly isolated Sca-1 cells were seeded into the upper chamber at a density of 4x104 cells per well. Subsequently, HUVEC and freshly isolated Sca-1 negative cells were seeded in parallel. Cells were then exposed to different concentrations (0, 10, 25 or 50ng/ml) of BDNF (Santa Cruz Biotechnology, Santa Cruz, CA, USA), which was used as chemoattractant in the lower chamber. Serum-free medium served as negative control and complete medium as background reference. To determine fluorescence at the maximal migration rate, equal numbers of cells were directly seeded to wells of the lower chamber while only medium was added to the upper chamber. Following 2 hours of incubation at 37 and 5% CO2, the non-migrated cells were removed from the upper side of the membrane. The cells attached to the lower side of the membrane were retrieved using detachment buffer. Collectively the detached cells and those cells that migrated to the lower chamber were fluorescently labelled with CyQuant dye and measured at 485/530nm. The migrated cells were quantified using the following three variables. Cell migrationMean fluorescence of test Mean fluorescence of negative control 100 Mean fluorescence of maximum migration Freshly isolated Sca-1 cells were attached to microscopic slides using a cytocentrifuge (Shandon Cytospin 4, Thermo Scientific, Bremen, Germany) at 100g for 8 minutes and afterwards washed with 1x phosphate buffered saline (PBS) to remove non-adherent cells. For cultured cells, Sca-1 cells were allowed to grow on glass coverslips until they reached 70% confluence and subsequently washed with PBS. The adherent cells were then fixed with 4% paraformaldehyde for 10 minutes. After washing with PBS, cells were permeabilized using 4% fetal bovine serum (FBS) and 0.2% Triton in PBS for 30 minutes at room MCE Chemical Eliglustat (hemitartrate) temperature. The cells were then incubated with the primary antibody against TrkB (1:100, Abcam, Cambridge, UK) in 4% FBS and 0.2% Triton in PBS overnight at 4. The10712926 untreated control was processed in parallel. Cells were washed thoroughly before incubation with the corresponding secondary antibody (goat anti-rabbit Alexa Fluor 488, 1:200 dilution, Life Technologies GmbH, Darmstadt, Germany) for 1 hour at room temperature. Nuclear staining was achieved using DAPI stain (1:100000, Roth, Karlsruhe, Germany). The slides with freshly isolated cells were then visualized using a microscope at 40x magnification while cultured Sca-1 cells were examined at 20x magnification.