Environmental Engineering Reference
In-Depth Information
fluorochrome-conjugated antibody at a dilution specified in the manufacturer
s
protocol. Cells were then washed twice with 1 mL Cell Wash Solution
(BD Biosciences, San Jose, CA, USA) each and resuspended in 500
'
L of the
same solution for further analysis on a four-color capable FACSCalibur (Becton-
Dickinson) flow-cytometer. Conjugated antibodies utilized included FITC-
conjugated CD90 (BD Pharmingen
ʼ
), CD44, and Her2 (Abcam), and
PE-conjugated CD29 and CD73. Fluorochrome-conjugated antibodies were pur-
chased from BD Pharmingen
™
unless otherwise specified. Acquisition and data
analyses were performed using CellQuest Pro software (Becton-Dickinson).
Immunocytochemistry
/
Immunofluorescence
: immunohistochemistry was perfor-
med for MSCs and SK-BR-3 tumor cell lines. Cells prepared for these analyses
were grown in 4-well glass chamber slides, were left to adhere for 24 h, and
colloidal suspensions of different concentrations were added. Untreated cells
were used as control. 72 h after addition of colloidal suspensions, medium was
removed, cells were washed, fixed with 4 % paraformaldehyde and permeabilized
with 0.1 % Triton X-100 and then investigated for expression of the proteins of
interest, using for labeling the following antibodies: monoclonal mouse anti-swine
Vimentin (clone V9), anti-human smooth muscle actin/HRP (clone 1A4), mono-
clonal anti-human endoglin, CD105 (clone SN6h), and polyclonal rabbit anti-
human c-erb-2 oncoprotein, respectively. All primary antibodies were provided
by DakoCytomation (Glostrup, Denmark) and tested for human specificity and
cross-reactivity. Staining protocol continued with secondary biotinylated antibody
binding, substrate addition (EnVision+
®
System-HRP DAB/AEC for use with
primary antibodies; Dako), and hematoxylin counterstaining of the nuclei following
the manufacturer
™
s procedures. Microscopy analysis was performed on a Nikon
Eclipse E800 microscope.
'
Conclusions
The influence of stable colloidal suspensions of magnetite nanoparticles
(prepared by combustion synthesis as well by co-precipitation route) on
tumor (SK-BR-3 breast cancer cell line) and normal (MSCs adult bone
marrow-derived mesenchymal stem cells) cell lines cultivated in vitro con-
ditions was investigated. As far as one can tell, this is the first evaluation of
the toxic effects of combustion synthesized magnetite nanoparticles.
Experimental results evidenced that combustion synthesized Fe
3
O
4
nanoparticles can be used and are compatible to in vitro culture conditions
of both normal and tumor cells. After 48 h in culture media, combustion
synthesized nanoparticles and co-precipitated nanoparticles decreased the
proliferation rate of normal MSCs and SK-BR-3 tumor cells. Viability of
normal MSCs treated with combustion synthesized nanoparticles was higher
than the viability of MSCs treated with co-precipitated nanoparticles. At the
same time, SK-BR-3 tumor cells treated with combustion synthesized
(continued)
