tissue: Umbilical Cord cell type: Mesenchymal stem and progenitor cells and Endothelial Colony Forming Cells
Treatment protocol
Identical MSPC/ECFC pairs from healthy newborn humans, passage number 1 under normal condition without pretreatment
Growth protocol
The MSPC and ECFC cells were isolated, purified and cultured for 7-10 days using alpha MEM and EGM-2 culture media enriched with human pooled lysate (HPL) respectively.
Extracted molecule
protein
Extraction protocol
In order to detect signaling proteins, plugs containing a total number of 32x106 cells (to allow for recovery of an appropriate protein amount based on previous titration) were explanted one day (24h) after implantation. Explants were homogenized using 400 µL Triton X-100 lysis buffer containing proteinase- and phosphatase-inhibitors (Roche, IN, USA) followed by magNAlyser centrifugation (700xg, 20 sec.), sonification (5x10 sec. with 10 sec. cooling steps in between;Imlab, Boutersem, Belgium) and ultra-centrifugation (100,000xg,30 min;BeckmanCoulterGmbH, Vienna, Austria). Protein concentrations were determined by a Bradford assay (Bio-Rad, CA, USA) and optical density (OD) was measured with a Spectramax instrument (Molecular Devices, Sunnyvale, CA, USA). Protein lysates were subjected to the KinexTM antibody microarray as a customized service (Kinexus Bioinformatics Corp., Vancouver, Canada, www.kinexus.ca) comparing different cellular compositions for protein expression alterations.
Label
Cy3
Label protocol
An amount of 50 µg of protein lysate from each sample was labeled with appropriate fluorescent dye followed by the non-specific fluorescent binding blockade. In order to load two samples in microarray fields side by side on the same array, an incubation chamber was mounted on to each microarray. Sample incubation and washing steps for removing unbound proteins was performed per manufacturer's protocol
Channel 2
Source name
Protein extraction from plugs containing ECFC only (N=3), all harvested from in vivo system after 24h.
Cultured ECFCs from umbilical cord, healthy newborn humans, passage number 1 under normal condition (alpha MEM medium enriched with HPL, 7-10 days in 37°C incubator without pretreatment.
Growth protocol
The ECFC cells were isolated, purified and cultured for 7-10 days using EGM-2 culture medium enriched with human pooled lysate (HPL).
Extracted molecule
protein
Extraction protocol
In order to detect signaling proteins, plugs containing a total number of 32x106 cells (to allow for recovery of an appropriate protein amount based on previous titration) were explanted one day (24h) after implantation. Explants were homogenized using 400 µL Triton X-100 lysis buffer containing proteinase- and phosphatase-inhibitors (Roche, IN, USA) followed by magNAlyser centrifugation (700xg, 20 sec.), sonification (5x10 sec. with 10 sec. cooling steps in between;Imlab, Boutersem, Belgium) and ultra-centrifugation (100,000xg,30 min;BeckmanCoulterGmbH, Vienna, Austria). Protein concentrations were determined by a Bradford assay (Bio-Rad, CA, USA) and optical density (OD) was measured with a Spectramax instrument (Molecular Devices, Sunnyvale, CA, USA). Protein lysates were subjected to the KinexTM antibody microarray as a customized service (Kinexus Bioinformatics Corp., Vancouver, Canada, www.kinexus.ca) comparing different cellular compositions for protein expression alterations.
Label
Cy5
Label protocol
An amount of 50 µg of protein lysate from each sample was labeled with appropriate fluorescent dye followed by the non-specific fluorescent binding blockade. In order to load two samples in microarray fields side by side on the same array, an incubation chamber was mounted on to each microarray. Sample incubation and washing steps for removing unbound proteins was performed per manufacturer's protocol
Hybridization protocol
Washing steps were completed using manufacturer's protocols.
Scan protocol
A pair of 16-bit images from each array was captured with a Perkin-Elmer ScanArray Reader laser array scanner (Waltham, MA, USA). Signal quantification was carried out with ImaGene 8.0 from BioDiscovery (El Segundo, CA, USA) using pre-determined settings for spot partitioning as well as background correction according to the manufacturer's protocol
Description
comparison of signaling protein expression in MSPC+ECFC combination plugs in vivo that leads in to vessel formation in later time point vs signaling protein expression in ECFC only plugs in vivo.
Data processing
Z normalization for the data was used since it displays greater stability as a result of examining where each signal falls in the overall distribution of values within a given sample rather than adjusting all of the signals in a sample by a single common value . The z-ratio value was used to quantify the signal intensity. Z scores were calculated by subtracting the overall average intensity of all spots within a sample from the raw intensity for each spot, and dividing it by the standard deviations (SD) of all of the measured intensities within each sample (Cheadle et al., 2003). Z-ratios were further calculated by taking the difference between the averages of the observed protein Z scores and dividing by the SD of all of the differences for that particular comparison.Calculated Z-ratios can be used in multiple comparisons without further reference to the individual conditional standard deviations by which they were derived. Z-ratio of equal to or more than ±1.2 was considered as a significantchange based on the core facility manufacturer’s instructions .
