Biological Membranes as Protein Aggregation Matrices and Targets of Amyloid Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 232

11. The cover slips were subsequently washed three times with
PBS, dried for 30 min at room temperature and mounted up-
side-down on microscope slides with a drop of Vectashield
gel mounting medium. Then the cover slips were enameled
around the edges to prevent a shift during confocal analysis.
12. Cell fluorescence was analyzed by a confocal Leica TCS SP5
scanning microscope using excitation lines at 488 nm and
568 nm for fluorescein and Texas Red, respectively. A series
of optical sections (1024 × 1024 pixels), 1.0 µm in thickness,
were taken through the cell depth for each examined sample
using a Leica Plan Apo 63× oil immersion objective and pro-
jected as a single composite image by superimposition. A vari-
able number of cells (ranging from 10 to 20) were estimated
on regions of interest in each experiment.

1. 5 × 10 5 human SH-SY5Y neuroblastoma cells were resuspended
in 300 µL culture medium and transferred to a FACS tube.
2. Cells were exposed to 3.0 µM Ab42-FAM ADDLs in culture
medium for different periods of time (0, 10, 30, 60 min) in a
5.0% CO 2 humidified atmosphere at 37°C under moderate
agitation (see Note 7).
3. Cells were analyzed by a FACSCanto (BD, San Jose, CA)
using the FACSDiva Software (BD, Milan, Italy).

3.3.2. Quantitation of Cell
Membrane-Bound ADDLs
by Flow Cytometric
Analysis

1. Human SH-SY5Y neuroblastoma cells were plated at a den-
sity of 5 × 10 4 per glass cover slip and incubated in culture
medium for 24 h in a six-well plate in a 5.0% CO 2 humidified
atmosphere at 37°C.
2. The cells were loaded with 2.0 mM calcein-AM (650 mL per
each cover slip in a six-well plate) for 20 min in a 5.0% CO 2
humidified atmosphere at 37°C.
3. Following several washes with PBS (see Note 9), the cells
were exposed to 1.0 µM ADDLs added to the culture medium
for different periods of time (0, 10, 30, 60 min) in a 5.0%
CO 2 humidified atmosphere at 37°C.
4. After several washes with PBS, the cells were fixed in 2% buff-
ered paraformaldehyde for 10 min at room temperature.
5. After washing three times with PBS, the cover slips were dried
for 30 min at room temperature and mounted up-side-down
on microscope slides with a drop of Vectashield gel mounting
medium. Then the cover slips were enameled around the
edges to prevent a cover slip shift during confocal analysis.
6. The decay of calcein fluorescence was analyzed by a confocal
Leica TCS SP5 scanning microscope equipped with an argon
laser source for fluorescence measurements at an excitation

3.3.3. Confocal Microscopy
Analysis of Plasma
Membrane
Permeabilisation