Page 143 - Physico-Chemical Niche Conditions for Bone Cells
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2D-images using CellProfiler software (Broad Institute of Harvard and MIT, Cambridge, MA). To quantify the shape of the cell surface, elongation (eccentricity, equals 1 for a straight line and 0 for a perfect circle), circularity (form factor, 2π ´ area/perimeter; equals 1 for a perfect circle) [3], and extent (approaches 1 for compact cells without protrusions) were quantified. Cell volume was measured from single cell z-stacks using Medical Imaging Interaction Toolkit software (MITK; German Cancer Research Center, Heidelberg, Germany).
Table 1: primer sequences used for real-time PCR Target
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Chapter 6
    gene
PXNa       Forward     5’-CAGTCCGCAGCGAGTCA -3’
Primer sequence
 Reverse PXNb Forward Reverse ITGA5 Forward Reverse Ki67 Forward Reverse CCND1 Forward Reverse RUNX2 Forward Reverse OPN Forward Reverse COL1a1 Forward Reverse ALP Forward Reverse HPRT1 Forward Reverse
5’-CCTGGGCCATGAACTTGAAATC-3’ 5’-ACCAGGGAGAGATGAGCAGT-3’ 5’-AGGCCCTGCATCTTGAAATCT-3’ 5’-GGAAGGGACGGAGTCAGTG-3’ 5’-TAGACAGCACCACCTTGCAG -3’ 5’-CCCTCAGCAAGCCTGAGAA -3’ 5’-AGAGGCGTATTAGGAGGCAAG-3’ 5’-TCAAGTGTGACCCGGACTG-3’ 5’-GACTCCAGAAGGGCTTCAA TCT -3’ 5’-ATTACAGATCCCAGGCAGGC-3’ 5’-TCTGGCTCAGATAGGAGGGG-3’ 5’-CCCGGTGAAAGTGACTGATT-3’ 5’-TTCTTCAGAGGACACAGCATTC-3’ 5’-AACTGGTACATCAGCCCGAA-3’ 5’-TTCCGTACTCGAACGGGAAT-3’ 5’-GGAACAACCTGACTGACCCT -3’ 5’-CCTCCTTCCACCAGCAAGAA-3’ 5’-CCTAAGATGAGCGCAAGTTGAA-3’ 5’-CCACAGGACTAGAACACCTGCTAA -3’
 Abbreviations: ALP, alkaline phosphatase; COL1a1, collagen type I a1 chain; HPRT1, hypoxanthine phosphoribosyltransferase 1; PXNa, paxillin transcript variant a; PXNβ, paxillin transcript variant β; ITGA5, integrin a5; CCND1, cyclin D1; RUNX2, runt-related transcription factor 2; OPN, osteopontin.
To analyze focal adhesions, summated z-projections of the lower 15 slices of single cell images were made using ImageJ. From the resulting 2D images, integrin a5 staining
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