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An IMPORTANT NOTICE at the end of this data sheet addresses availability, .. metrics, see the IC Package Thermal Metrics application report (SPRA). An integrated circuit or monolithic integrated circuit (also referred to as an IC, a chip, or a microchip) is a set of electronic circuits on one small. Get Free Download Ebook and Manual Reference. Teryx Manual · Hellenbrand Pm6 O Ic 2 0 Series Repair Service Manual User Guides Manual · Chevrolet Equinox Pontiac Torrent Automotive Repair Manual 1st Edition.
The number of viable cells was determined using the trypan blue exclusion method and the cells were eventually cryopreserved as described in a previous manuscript Baufeld and Vanselow, All the chemicals for cell culture were purchased from Biochrom Berlin, Germany unless stated otherwise.
Cell Viability and Apoptosis Analysis On the 8th day of culture, spent culture media were collected in a 1.
The detached cells were added to the above pelleted cells to ensure the inclusion of floating and attached cells into the analysis. After gentle mixing, the tubes were incubated in the dark for 15 min. The fluorescence signal was quantified from single cells 10, counts using a flow cytometer Gallios, Beckman-Coulter, Germany and the data were analyzed using the Kaluza-software Beckman-Coulter, Germany.
The Abundance of transcripts was normalized using TBP as a validated house-keeping gene under low oxygen conditions Baddela et al. Overnight hybridization of RNA samples and probes was carried out in a hybridization oven followed by acquisition of the gene expression signals using an Affymetrix Gene Chip Scanner Normalization and background reduction of gene expression was performed using the robust multichip average method. The acquired data were subsequently analyzed using the TAC 4.
Estimation of Estradiol and Progesterone Concentration Estradiol E2 and progesterone P4 concentrations were estimated in the spent media using a sensitive single antibody 3H-radioimmunoassay performed in a competitive mode.
The antibodies were raised in rabbit and purified using affinity chromatography. Determination of Cell Proliferation Cell proliferation was analyzed by identifying the number of cells in different phases of the cell cycle using flow cytometer analysis.
GC were cultured for 8 days as described above. On the 8th day, spent culture media were collected in a 1. The detached cells were added to the floating cells of the corresponding wells. The data were subsequently analyzed using the Multicycle software Phoenix, United States.
Sequencing of PCR products was performed at the institutional core facility. Bioinformatics and Statistical Analysis All bioinformatic analyses were carried out for the human homologs of DE genes. The enriched gene ontology terms were recognized using WebGestalt, a WEB based gene set analysis tool kit. The canonical pathways and upstream regulators were identified using the Ingenuity pathway analysis tool IPA, Qiagen, Hilden.
Further, hub genes were recognized by constructing a protein-protein interaction network using NetworkAnalyst tool available at www. Microarray data analysis was performed using integrated statistical measures available in TAC 4.
Results Effect of Low Oxygen Levels on the Viability and Steroidogenesis of Granulosa Cells After subjecting GC to low and normal oxygen levels Figure 1 and Supplementary Figure S1 , the percentage of live, apoptotic and dead cells was determined using flow cytometric analysis by adding propidium iodide PI and annexin-V reagents to the detached cells.
However, unlike the viability status of the cells, levels of estradiol and progesterone were significantly reduced at low oxygen levels Figure 1D. Effect of low oxygen levels on the viability and steroidogenic capacity of granulosa cells. A,B Visualize representative histograms of cells treated with normal and low oxygen levels, respectively, in flow cytometry analysis.
Subsequent principal component analysis PCA of the microarray data sets Figure 2A showed that the samples from low and normal oxygen treatments were located most distant from each other with a variation of Further, a mere variation of 6.
Principle component analysis and clustered heat mapping. Unsupervised principal component analysis visualizes differences in the transcriptomes of bovine GC cultured at normal and low oxygen conditions A.
Blue and red color dots denote individual samples from normal and low oxygen conditions, respectively. Each axis indicates the fraction of percentage of variation out of total mapped variation among samples. A total of publicly annotated gene clusters Supplementary Data Sheet S3 were identified in the microarray data.
Specifically, and genes were up- and down-regulated, respectively, at low oxygen levels. The abundance of DE genes was visualized in the form of an interactive heat map with zoom in and out features , which was constructed using shinyHeatmaply package in R studio Supplementary Html File S1.
The same heat map is shown in a static form in Figure 2B. The top twenty down- and up- regulated genes at low oxygen levels are listed in Tables 1 , 2. Top twenty down-regulated genes at low oxic conditions. Top twenty up-regulated genes at low oxygen conditions.
However, their functions with respect to granulosa and luteal cell function is not yet known. Microarray data were validated using RNA samples isolated from three independent experiments, different from those used for microarray analysis. A total of six genes were selected for re-assessing the expression values.
The normalized expression of all these genes was found to be similar in both qPCR and microarray estimations Figure 3. Bioinformatics Interpretations All bioinformatic analyses were performed for the human homologs of DE genes. Initially, GO terms were generated separately for up- and down-regulated genes using the WebGestalt tool Figure 4.
This indicated the prioritized list of biological processes, cell components and molecular function categories for genes regulated by differential oxygen levels. Similar observations can also be noticed with respect to different molecular functions at different oxygen levels. Further, to understand the detailed functional changes induced by differential oxygen concentrations, enriched canonical pathways were identified using IPA.
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