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Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence
Article first published online: 13 AUG 2013
Published 2013 Wiley-Periodicals, Inc. This article is a US government work and, as such, is in the public domain in the United States of America.
Cytometry Part A
Volume 83, Issue 10, pages 962–972, October 2013
How to Cite
Zucker, R. M., Daniel, K. M., Massaro, E. J., Karafas, S. J., Degn, L. L. and Boyes, W. K. (2013), Detection of silver nanoparticles in cells by flow cytometry using light scatter and far-red fluorescence. Cytometry, 83: 962–972. doi: 10.1002/cyto.a.22342
- Issue published online: 20 SEP 2013
- Article first published online: 13 AUG 2013
- Manuscript Accepted: 28 JUN 2013
- Manuscript Revised: 18 JUN 2013
- Manuscript Received: 23 MAY 2012
- USEPA. Grant Numbers: EP09D000042, EP10D000713
Additional Supporting Information may be found in the online version of this article.
|cytoa22342-sup-0002-suppfig1.tif||997K||Supplemental Figure 1. Flow cytometry detection of 50 nm PVP-coated AgNP using SSC and fluorescence parameters. ARPE-19 cells were incubated with 3 to 100 µg/ml of AgNP for 24 hours. AgNP penetrated into cells increasing the side scatter in a dose-dependent manner. 0µg/ml (black), 3µg/ml (red), 10µg/ml (blue), 30µg/ml (green), and 100µg/ml (purple). The 3µg/ml lines in histograms of FL1 (530/3) and FL2 (585/42) were removed for figure clarity. The cells containing nanoparticles showed a small increase of fluorescence in FL1 (530/30), and FL2 (585/42) and a large increase FL3 (>670).|
|cytoa22342-sup-0003-suppfig2.tif||631K||Supplemental Figure 2. Relationship of side scatter and far red fluorescence of ARPE-19 cells treated with 75 nm AgNP for 24 hours. The side scatter increased more at low concentrations and leveled off at higher concentration, while the and far red fluorescence showed a small increase at low concentrations and a larger increase at higher concentrations. This may be related to clumping of these coated particles at higher concentrations of AgNP, as indicated in the morphology figures (Figure 4, Supplemental Figure 3).|
|cytoa22342-sup-0004-suppfig3.tif||6468K||Supplemental Figure 3. Comparison of dark field and fluorescence of 10 nm (left) and 75 nm (right) Ag nanoparticles. ARPE-19 cells incubated in slide chambers for 24 hours with (30 µg/ml) nano-Ag-PVP. Cells were fixed as described in Figure 3. Note the increased AgNP uptake with the 75 nm compared to the 10 nm observed by dark-field microscopy Magnification 600x|
|cytoa22342-sup-0005-supptable1.tif||259K||Supplemental Table 1. Data from histograms correlates to Figure 1. SSC and far red fluorescence changes after exposure of ARPE-19 cells to 1, 3, 10, 15, and 20 µg/mL of 75 nm PVP-coated AgNP for 24 hours. Side scatter showed a dose dependent increase of 4 fold while the fluorescence increase was more than ten times the scatter (about 40 fold over background).|
|cytoa22342-sup-0006-supptable2.tif||326K||Supplemental Table 2. Changes in SSC and fluorescence of ARPE-19 cells after 24 h exposure to 0, 3, 10, 30, and 100 µg/mL of 50 nm PVP-coated AgNP. The data correlate to the histograms in Figure 1. All values are normalized to the untreated controls. There was minimum change in the intensity in FL1 (530/30) and FL2 (585/42) parameters compared to the far red fluorescence detected in PMT Fl3 (>670 nm).|
|cytoa22342-sup-0007-supptable3.tif||367K||Supplemental Table 3. Comparison of 3 sizes of PVP and citrate coated AgNP at a concentration of 30 µg/ml. Both citrate and PVP coated AgNP show an increase in far red fluorescence with the 50 nm and 75 nm Ag nanoparticles. Minimal changes in all fluorescence channels are observed with both coatings using 10nm particles|
|cytoa22342-sup-0008-supptable4.tif||359K||Supplemental Table 4. A comparison of 3 flow cytometers (2 FACSCalibur and Attune) were used to demonstrate a similar increase in SSC and far red fluorescence using cells exposed to different doses between 1 and 20 µg/ml of 75 nm PVP-Ag NP for 24 hours.|
|cytoa22342-sup-0009-supptable5.tif||404K||Supplemental Table 5. LSRII: Induction of fluorescence by AgNP (20 µg/ml) for 24 hours expressed as a ratio of treated/control. There was a decrease in FSC and an increase in SSC indicating the AgNP entered the cells. The maximum increase was observed in the Cy7 channels from the 488 nm and 633 nm lasers. The increase in fluorescence from the 488 nm laser was detected in all 5 PMTs and 2 side scatter channels. The 633 nm red laser fluorescence was detected in the 3 PMTs while the 405 nm laser fluorescence was detected in 2 PMTs. The table shows the name of the PMT filter and bandpass range in front of the PMT that was used to detect fluorescence. Side scatter (SSC) is shown to illustrate the magnitude of change in the cells that acquired AgNP compared to changes observed in fluorescence.|
|cytoa22342-sup-0010-supptable6.tif||334K||Supplemental Table 6. ARPE-19 cells were exposed to three different sizes and types of TiO2 NP to (0, 3, 10 µg/ml) for 24 hours. The data show that there was minimal increase in the far red fluorescence from the cells treated with TiO2NP indicating that TiO2NP reacts differently to the cells compared to AgNP.|
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