Gene Expression Analysis of Paclitaxel-Treated HT29 Cells Using the xCELLigence™ System and RealTime ready™ Panels

Scheuermann, T.; Sun, Yi; Sagner, Susanne; Rueger, Barbara

doi:10.2144/000113201

Cited by 4 publications

(3 citation statements)

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“…We investigated the effect of SPARC deficiency on HTF proliferation compared against treatment with MMC, which is well‐established for its anti‐proliferative activity. The real‐time cell analyser SP instrument [20] was used to analyse HTF cell growth. Cell index values produced by the MMC‐treated cells indicated the lack of proliferation while the cell index values of the untreated control cells increased progressively with time (Fig.…”

Section: Resultsmentioning

confidence: 99%

In vitro analyses of the anti‐fibrotic effect of SPARC silencing in human Tenon’s fibroblasts: comparisons with mitomycin C

Seet

Toh

et al. 2012

J Cellular Molecular Medi

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Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon’s fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β2 were also reduced. Importantly, TGF-β2 failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics.

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Section: Resultsmentioning

confidence: 99%

In vitro analyses of the anti‐fibrotic effect of SPARC silencing in human Tenon’s fibroblasts: comparisons with mitomycin C

Seet

Toh

et al. 2012

J Cellular Molecular Medi

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“…The RTCA SP instrument was used to analyze cell growth. 21 Cell index values produced by the shRNA1-transfected cells indicated the lack of proliferation while the cell index values of the control transfected cells increased progressively with time suggesting normal growth proliferation (Fig. 3A).…”

Section: Depletion Of Slc4a11 Reduced Hcec Proliferationmentioning

confidence: 94%

“…A real time cell analyzer (RTCA) (xCelligence RTCA SP; Roche Diagnostics GmbH, Penzberg, Germany) was used to assess cell proliferation according to manufacturer's instructions. 21 HCECs were seeded onto the RTCA's E-96 plate 5 days after transfection in quadruplicates. The plated cells were allowed to equilibrate for at least 30 minutes in the tissue culture incubator before electrode resistance was recorded.…”

Section: Real-time Cell Growth Measurementmentioning

confidence: 99%

Depletion ofSLC4A11Causes Cell Death by Apoptosis in an Immortalized Human Corneal Endothelial Cell Line

Liu

Seet

Koh

et al. 2012

Invest. Ophthalmol. Vis. Sci.

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Optimization of cytotoxicity assay by real-time, impedance-based cell analysis

Ramis

Martı́nez-Alarcón

Quereda

et al. 2013

Biomed Microdevices

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This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.

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Gene Expression Analysis of Paclitaxel-Treated HT29 Cells Using the xCELLigence™ System and RealTime ready™ Panels

Cited by 4 publications

References 0 publications

In vitro analyses of the anti‐fibrotic effect of SPARC silencing in human Tenon’s fibroblasts: comparisons with mitomycin C

In vitro analyses of the anti‐fibrotic effect of SPARC silencing in human Tenon’s fibroblasts: comparisons with mitomycin C

Depletion ofSLC4A11Causes Cell Death by Apoptosis in an Immortalized Human Corneal Endothelial Cell Line

Optimization of cytotoxicity assay by real-time, impedance-based cell analysis

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