Environmental Engineering Reference
In-Depth Information
Pen/Strep solution 10,000 IU/mL (PromoCell) in an incubator, at 37
Cina
humidified and 5 % CO
2
atmosphere. Cells were seeded at 10,000 cells/cm
2
in
appropriate well plates and allowed to attach for 24 h previous to MNPs addition.
Bone marrow samples were obtained after signing the written informed consent
elaborated under an approved protocol by the Ethics Committee of “Victor Babe
ș
”
University of Medicine and Pharmacy Timi
ș
oara, according to the World Medical
Association Declaration of Helsinki.
Cells scanning electron microscopy
(
SEM
): scanning electron microscopy was
performed for identification of morphological changes in MSCs and SK-BR-3 cell
lines induced by the colloidal suspensions. Cells were cultured at 7,000 cells/cm
2
in
24-well format cell culture inserts (BD Labware Europe, Le Pont De Claix, France)
and colloidal suspensions were added 48 h after colloidal suspensions addition,
cells were pre-fixed for 1 h with 2.5 % buffered glutaraldehyde (in PBS), rinsed
three times in PBS, and the 0.4
m pore-sized membranes were detached from the
culture inserts. For better image quality, cells fixed on the membranes were sputter-
coated with platinum-palladium and examined with a FEI Quanta 3D FEG electron
microscope
ʼ
(FEI Company, Eindhoven, NL) generating digital
electron
micrographs.
Cells transmission electron microscopy
(
TEM
): MSCs and SK-BR-3 tumor cells
were employed for investigation of ultrastructural details changes, 72 h after
addition of colloidal suspensions in different concentrations. Cells were prefixed
for 1 h with glutaraldehyde (2.5 % in PBS), rinsed three times in PBS, and post-
fixed for 1 h in osmium acid (2 % in PBS). Dehydration was done in graded acetone
in distilled water dilutions, followed by infiltration with Epon resin. Sections of
about 100 nm, obtained on a diamond knife (Diatome) with Leica UC6 ultrami-
crotome were post-stained with lead citrate and uranyl acetate. The grids were
examined with a FEI Tecnai 12 transmission electron microscope (FEI Company).
Annexin V
/
PI assay
: because MTT viability assay is based on spectrophotomet-
ric detection of color change due to active substrate metabolization in live cells, and
the presence of suspended Fe
3
O
4
nanoparticles can influence color detection, we
used Annexin V-FITC/PI viability assay, which is based on fluorescence emission
and is performed on flowcytometer, thus resulting in less ambiguities of results.
Annexin V-FITC (Miltenyi Biotec, Gladbach, Germany) was used in cell death
flowcytometric studies (apoptosis) combined with Propidium Iodide Staining Solu-
tion (BD Biosciences, San Jose, CA, USA) following the manufacturer
s protocol.
'
Shortly, 10
6
cells were washed in 1
Annexin V Binding Buffer (BD Pharmigen)
and centrifuged at 300
g
for 10 min, resuspended in the same solution and
incubated with 10
L of Annexin V-FITC for 15 min in the dark. After washing
the cells with 1 mL specific binding buffer and centrifugation, the cell pellet was
resuspended in 500
ʼ
ʼ
L binding buffer and 1
ʼ
g/mL of PI solution was added
immediately prior to analysis by flowcytometry.
Flowcytometric analysis
: MSCs and SK-BR-3 cells in culture 80-90 % conflu-
ence were detached using 0.25 % Trypsin-EDTA (Sigma), washed two times
with PBS, resuspended in 100
L PBS at a concentration of 10
5
cells/mL and
incubated in the dark at room temperature for 30 min with mouse anti-human
ʼ
