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Single-walled carbon nanotube interactions with HeLa cells.

Yehia HN, Draper RK, Mikoryak C, Walker EK, Bajaj P, Musselman IH, Daigrepont MC, Dieckmann GR, Pantano P - J Nanobiotechnology (2007)

Bottom Line: Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACT
This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

No MeSH data available.


Flow cytometry analysis of intracellular MitoSOX™ Red fluorescence from live HeLa cells incubated at 37°C for 60 h in: (A) DMEM/FBS, (B) CoMoCAT DM-SWNTs, (C) DMEM/FBS + MitoSOX™ Red, (D) DM-SWNTs + MitoSOX™ Red, and (E) DMEM/FBS + MitoSOX™ Red + H2O2. The x-axis denotes the MitoSOX™ Red fluorescence detected in the 564–606 nm spectral region and the y-axis denotes the number of events recorded for each analysis.
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Figure 12: Flow cytometry analysis of intracellular MitoSOX™ Red fluorescence from live HeLa cells incubated at 37°C for 60 h in: (A) DMEM/FBS, (B) CoMoCAT DM-SWNTs, (C) DMEM/FBS + MitoSOX™ Red, (D) DM-SWNTs + MitoSOX™ Red, and (E) DMEM/FBS + MitoSOX™ Red + H2O2. The x-axis denotes the MitoSOX™ Red fluorescence detected in the 564–606 nm spectral region and the y-axis denotes the number of events recorded for each analysis.

Mentions: In each fluorescence-based flow cytometry experiment, six different cell samples/controls were prepared and analyzed in triplicate with each individual trial representing the analysis of thousands of cells. Fluorescence microscopy was also used to validate that MitoSOX™ Red was distributed throughout the cytoplasms of cells, and that negligible dye leaked from the cells (data not shown). The first two flow cytometry control experiments involved measuring responses of cells incubated in DMEM/FBS without MitoSOX™ Red (± DM-SWNTs). These dye-free controls were prepared to establish background fluorescence levels of unstained HeLa cells (± DM-SWNTs) and are represented in the plot of events vs. MitoSOX™ Red fluorescence intensities as shown in Figures 12A &12B (and Figures S6A&B in Additional File 4). The means and standard deviations of fluorescence intensities from these two control experiments without MitoSOX™ Red were 3.07 ± 0.15 and 2.40 ± 0.44 a.u. for DMEM/FBS and DM-SWNT treated cells, respectively. Next, since it has recently been reported that binding of fluorescent viability dyes to CNTs can add uncertainty to cytotoxicity assessments [65], our series of experiments also included a comparison of responses from positive controls ± DM-SWNTs. Specifically, the responses of cells loaded with MitoSOX™ Red and exposed to 5 μmoles hydrogen peroxide were analyzed in the presence and absence of DM-SWNTs (Additional File 4; Figures S7A and S7B respectively). Both samples possessed statistically-similar fluorescence intensities indicating that SWNT quenching of the MitoSOX™ Red fluorescence was minimal.


Single-walled carbon nanotube interactions with HeLa cells.

Yehia HN, Draper RK, Mikoryak C, Walker EK, Bajaj P, Musselman IH, Daigrepont MC, Dieckmann GR, Pantano P - J Nanobiotechnology (2007)

Flow cytometry analysis of intracellular MitoSOX™ Red fluorescence from live HeLa cells incubated at 37°C for 60 h in: (A) DMEM/FBS, (B) CoMoCAT DM-SWNTs, (C) DMEM/FBS + MitoSOX™ Red, (D) DM-SWNTs + MitoSOX™ Red, and (E) DMEM/FBS + MitoSOX™ Red + H2O2. The x-axis denotes the MitoSOX™ Red fluorescence detected in the 564–606 nm spectral region and the y-axis denotes the number of events recorded for each analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2131758&req=5

Figure 12: Flow cytometry analysis of intracellular MitoSOX™ Red fluorescence from live HeLa cells incubated at 37°C for 60 h in: (A) DMEM/FBS, (B) CoMoCAT DM-SWNTs, (C) DMEM/FBS + MitoSOX™ Red, (D) DM-SWNTs + MitoSOX™ Red, and (E) DMEM/FBS + MitoSOX™ Red + H2O2. The x-axis denotes the MitoSOX™ Red fluorescence detected in the 564–606 nm spectral region and the y-axis denotes the number of events recorded for each analysis.
Mentions: In each fluorescence-based flow cytometry experiment, six different cell samples/controls were prepared and analyzed in triplicate with each individual trial representing the analysis of thousands of cells. Fluorescence microscopy was also used to validate that MitoSOX™ Red was distributed throughout the cytoplasms of cells, and that negligible dye leaked from the cells (data not shown). The first two flow cytometry control experiments involved measuring responses of cells incubated in DMEM/FBS without MitoSOX™ Red (± DM-SWNTs). These dye-free controls were prepared to establish background fluorescence levels of unstained HeLa cells (± DM-SWNTs) and are represented in the plot of events vs. MitoSOX™ Red fluorescence intensities as shown in Figures 12A &12B (and Figures S6A&B in Additional File 4). The means and standard deviations of fluorescence intensities from these two control experiments without MitoSOX™ Red were 3.07 ± 0.15 and 2.40 ± 0.44 a.u. for DMEM/FBS and DM-SWNT treated cells, respectively. Next, since it has recently been reported that binding of fluorescent viability dyes to CNTs can add uncertainty to cytotoxicity assessments [65], our series of experiments also included a comparison of responses from positive controls ± DM-SWNTs. Specifically, the responses of cells loaded with MitoSOX™ Red and exposed to 5 μmoles hydrogen peroxide were analyzed in the presence and absence of DM-SWNTs (Additional File 4; Figures S7A and S7B respectively). Both samples possessed statistically-similar fluorescence intensities indicating that SWNT quenching of the MitoSOX™ Red fluorescence was minimal.

Bottom Line: Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACT
This work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

No MeSH data available.