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In-Depth Information
NH
4
Cl for 10 min. Three rinses were fi nally made with phosphate-buffered
saline (PBS) (provided by Lonza, Belgium). The nonspecifi c sites were blocked
using 3% BSA solution (Sigma Aldrich, France) for 30 min. Then, a primary
antibody anti-TrkB was added (at a concentration of 10
g/ml) to the fi xed
cells for 1 h in a humid chamber at 37°C. After three successive washes with
a solution of 1% BSA, the cells were incubated with a secondary antibody
(diluted at 1/500), in the dark for 45 min, and then washed three times with
PBS. The recovered strips were mounted on microscope slides using a drop of
Vectashield mounting liquid that preserves the fl uorescence of the secondary
antibody. The samples were sealed with varnish to prevent premature drying
of the preparation and stored at 4°C in the dark.
The imaging was performed on the day of the sample preparation using a
confocal microscope ZEISS (model LSM 510, Zeiss, Germany). The experi-
ments were set with a fl uorescence excitation wavelength of 633 nm. Images
were recorded with fl uorescence emission collected from 650 nm. Differential
interference contrast (DIC), or Nomarski type, images were obtained in paral-
lel. An oil objective 63
μ
×
/1.4 was employed.
11.3.3.3 Cellular Viability Assessed by MTT Tests
The cells viability
was determined based on the activity of the mitochondrial dehydrogenase
enzyme that cleaves the tetrazolium salt 3 - [4,5 - dimethylthiazol] - 2,5 -
diphenyltetrazolium (MTT) into a formazan product. The cells were seeded
in 96-well plates and grown to 80% confl uence in culture medium. For dif-
ferentiation, they were incubated for 5 days after adding, in each well, a
volume of 200
M) prepared in culture medium.
Two washes were then accomplished with serum-free DMEM before replacing
it with DMEM (without serum) containing increasing concentrations of lipid
formulations and/or BDNF (2 ng/ml). Every experimental condition was
carried out on 8 wells that constituted one column of the 96-well plate. After
72 h of incubation, 20
μ
l retinoic acid solution (10
μ
l of MTT (5 mg/ml), prepared in PBS, were added to
each well, with the exception for the control wells. Two hours later, the medium
was aspirated. The cells and the formazan crystals were solubilized by adding
200
μ
l of dimethyl sulfoxide (DMSO). The plate was stirred for a few minutes
and the optical density was determined using a plate reader operated at a
wavelength 570 nm. The results are presented in percentages of cell viability
compared to controls.
μ
11.4 PHYSICOCHEMICAL AND BIOLOGICAL INVESTIGATIONS OF
NANOPARTICULATE MIXED LIPID SYSTEMS
11.4.1 Nanoparticles Prepared from MO/DOPE-PEG
2000
/OA/EPA
Self-Assembled Lipid Mixtures
The fi rst objective of the study was to determine the molar ratios between the
investigated lipids favoring the formation of multicompartment nanoparticles
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