Material and Methods 16 rene surfaces. 1,000 cells/cm 2 were seeded in triplicates into 6-well dishes after coating with Col1, FN or LN as described earlier in cell volume measurement (2.2.4.). FBS concentration was reduced to 5% to avoid additional effects from proteins within the serum. Normoxic time-lapse was captured for 72h with a AxioVert S100 (Zeiss, Jena, Germany) inverted microscope and an automated microscopy table (SCAN IM 130x100 - 1mm; Märtzhäuser-Wetzlar GmbH & Co. KG, Steindorf, Germany) and a humidified, heated microscope climate chamber (Incubator XL S, Pecon, Erbach, Germany). Hypoxic time-lapse was realised with a self-designed crystal clear polymethylmetaacrylate (PMMA) box for the 6-well dish. The box was completely magnetic sealed. While temperature was controlled by the standard climate chamber, oxygen and carbon dioxide were controlled with a ProOx 110 and ProCO2 gas feedback devises (both from BioSpherix, New York, USA). Acquired data was manually analyzed with the plugin MTrackJ for the open-source software ImageJ (National Institutes of Health of the United States, http://rsb.info.nih.gov/ij/index.html). At least 100 cells per ECM coating were evaluated for their velocity and Euclidean distance. All cells within the field of view (FOV) were included and dividing cells were marked separately. Experiments were repeated twice. In sum 1019 cell tracks with over 105,000 data points were evaluated manually. 2.2.6. RT-PCR To detect possible differences of the integrin expression, ribonucleic acid (RNA) from both groups was isolated in passage 3 by an RNeasy ® Mini Kit (Qiagen, Hilden, Germany) according to the manufactures protocol. In brief, cell layer was washed with PBS, subsequently incubated with RLT buffer, supplemented with 1% βmercaptoethanol (Merck, Darmstadt, Germany) and cell lysate was detached with a cell scraper (Sarstedt, Nümbrecht, Germany). RNA was then purified with RNeasy ® spin columns according to the company’s protocol and frozen at -20°C until synthesizing of complementary deoxyribonucleic acid (cDNA). Prior RT-PCR integrin screening, hexamer-primed reverse transcription was performed using a cloned AMV first strand cDNA synthesis kit (Invitrogen, Karlsruhe, Germany) in a thermocycler (DNA Engine ® , Bio-Rad, Munich, Germany). Table 3 shows a summary of all used integrin primer sequences. Samples were normalized against a house keeping gene GAPDH prior PCR product analysis within 1,5% agarose gel (Biozym, Hessisch
Material and Methods 17 Oldendorf, Germany) and imaged in a UV gel imager (Infinity-1000, Vilber Lourmat, Eberhardzell, Germany). Tab. 3: List of used primer sequences (F: forward, R: reverse, bp: base pairs). gene primer sequence GAPDH α1 α2 α3 α5 α6 α11 αV β1 β3 β5 F 5’-ACATCAGCCAAGTCAATGTTTCG-3’ R 5’-AGCATTAACAGCAACAATCCGG-3’ F 5’-GCTGCTGTGCATTAGATATTAG-3’ R 5’-CTGTAACTTCTGGTGAAATCCT-3’ F 5’-ATCTTGAGAGCCACAGTCA-3’ R 5’-CTGGGTCCTTCTTTCTAGTTC-3’ F 5’-ATCTTGAGAGCCACAGTCA-3’ R 5’-CTGGGTCCTTCTTTCTAGTTC-3’ F 5’-ACTAGGAAATCCATTCACAGTTC-3’ R 5’-GCATAGTTAGTGTTCTTTGTTGG-3’ F 5’-CTTGGAGAAGATGGGTTTATT-3’ R 5’-GAATACAGATAGGGGAGGAAA-3’ F 5’-TGGGCGCACCCATGTACTTC-3’ R 5’-ATGGCTCCTGCGTGGTTGTC-3’ F 5’-GGAGCACATTTAGTTGAGGTAT-3’ R 5’-ACTGTTGCTAGGTGGTAAAACT-3’ F 5’-CTGCTGTAGACATTTGCTATGA-3’ R 5’-AAAACACCAGCAGCCGTGTAAC-3’ F 5’-CTGCTGTAGACATTTGCTATGA-3’ R 5’-GCCAAGAGGTAGAAGGTAAATA-3’ F 5’-GTATGCTGGTTTTACAGACTCC-3’ R 5’-TGCCCTTTTGTAGCCTCCTTG-3’ 2.3. Statistical analysis product size (bp) 181 50 241 51 217 48 201 52 201 52 213 48 223 55 274 46 322 52 211 52 407 54 annealing temp. [°C] Statistical analysis was performed using GraphPad Prism version 5.0 (Statcon, Witzenhausen, Germany). The comparison between different groups was done using either a Mann-Whitney u-test for non-Gaussian distributions or for Gaussian distributions a Student’s t-test for equal variances or Welch-test for unequal variances, respectively. A value of p < 0.05 was considered significant. Data is presented as mean ± standard deviation. All experiments were repeated at least twice.
Acknowledgments 67 Acknowledgments