The C166 endothelial cell line (ATCC, Rockville, MD) was cultured in DMEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin—streptomycin (Sigma–Aldrich, St. Louis, MO). Cells were grown to confluency and then treated with medium only or with prednisolone (Sigma–Aldrich; 100–300 ng/ml). Following 6 hr of exposure, total RNA was isolated using a commercial kit (Qiagen RNeasy kit, Valencia, CA) according to the manufacturer’s instructions. RNA was reverse transcribed into cDNA as described previously [Skeie et al., 2010 (link)]. Quantification of specific mRNA was performed by SYBR Green real-time PCR using an ABI Prim 7700 Sequence Detection System (Applied Biosystems) [Mullins et al., 2006 ]. Triplicate reactions were performed for each sample. For some experiments, testosterone (Sigma–Aldrich; 100 and 10 ng/ml) was included to assess the role of a corticosteroid without known impact on CSC in humans.
Abi prim 7700 sequence detection system
Manufactured by Thermo Fisher Scientific
The ABI Prim 7700 Sequence Detection System is a real-time PCR instrument designed for gene expression analysis, genotyping, and other quantitative PCR applications. The system utilizes a thermal cycler, fluorescence detection, and specialized software to accurately measure and analyze nucleic acid sequences in real-time.
Automatically generated - may contain errors
Lab products found in correlation
Prednisolone, by Merck Group (2 mentions)
C166 endothelial cell line, by Merck Group (2 mentions)
Penicillin streptomycin, by Merck Group (2 mentions)
Testosterone, by Merck Group (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Qiamp rna blood mini kit, by Qiagen (1 mentions)
Abi prism 7900 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase, by Qiagen (1 mentions)
3 protocols using abi prim 7700 sequence detection system
1
Endothelial Cell Response to Prednisolone
2
RNA Isolation and Gene Expression Analysis
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Total RNA was isolated from white blood cells using the QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany) supplemented with RNase-free DNase (Qiagen) and stored at minus 80°C [5] (link), [15] (link). For reverse transcription (RT) of RNA we used 300 ng of total RNA in a final volume of 30 µl and added adequate amounts of TaqMan Reverse Transcription Reagents (Applied Biosystems, Foster City, USA). The quantification of Cε germline transcripts we used the ABI Prim 7700 Sequence Detection System (Applied Biosystems) and the following primers: forward 5′-ACAGGCACCAAATGGACGAC-3′ , reverse 5′-TTGCAGCAGCGGGTCAA-3′ . The minor groove binding probe had the sequence 5′-CACAGAGCCCATCCG-3′ . The quantification of the other genes was performed on an ABI Prism 7900 Sequence Detection System (Applied Biosystems) using the TaqMan low density array (LDA) system of Applied Biosystems. The determined gene expression values were normalized to the parallel measured endogenous controls 18S rRNA. We analyzed the data with the comparative Ct method according to the manufacturer’s instructions (Applied Biosystem). Tests regarding RNA stability were performed [44] (link).
3
Endothelial Cell Prednisolone Response
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The C166 endothelial cell line (ATCC, Rockville, MD) was cultured in DMEM supplemented with 5% fetal bovine serum (FBS) and 1% penicillin—streptomycin (Sigma–Aldrich, St. Louis, MO). Cells were grown to confluency and then treated with medium only or with prednisolone (Sigma–Aldrich; 100–300 ng/ml). Following 6 hr of exposure, total RNA was isolated using a commercial kit (Qiagen RNeasy kit, Valencia, CA) according to the manufacturer's instructions. RNA was reverse transcribed into cDNA as described previously Skeie et al., 2010 (link). Quantification of specific mRNA was performed by SYBR Green real-time PCR using an ABI Prim 7700 Sequence Detection System (Applied Biosystems) Mullins et al., 2006 . Triplicate reactions were performed for each sample. For some experiments, testosterone (Sigma–Aldrich; 100 and 10 ng/ml) was included to assess the role of a corticosteroid without known impact on CSC in humans.
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