Biological Membranes as Protein Aggregation Matrices and Targets of Amyloid Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 224

(FBS, Sigma), 1.0% glutamine, and 1.0% antibiotics (60 mg/
ml penicillin and 100 mg/ml streptomycin).
2. Solutions of trypsin (0.25%) and phosphate-buffered saline
(PBS) are added to the culture dishes when required.
3. Microscope cover slips (20 × 20 × 0.25 mm) and microscope
slides (25 × 75 × 1 mm).
4. Buffered paraformaldehyde (Sigma): 2.0% solution in PBS
and store at 4°C.
5. Fluorescein-conjugated wheat germ agglutinin (Molecular
Probes, Eugene, OR): 5.0 mg/ml solution in PBS; store at
−20°C (see Note 6).
6. Glycerol solution: 3.0% (v/v) glycerol solution in PBS with
0.5% BSA (prepare fresh for each experiment).
7. Primary antibody: Mouse monoclonal 6E10 anti-Ab anti-
body (Signet, Dedham, MA, USA); store at −20°C.
8. PBS with 1% fetal bovine serum (FBS).
9. Secondary antibody: Anti-mouse IgG conjugated to Texas Red
(Vector Laboratories, DBA, Italy); store at 4°C (see Note 6).
10. Calcein-AM (Molecular Probes, Eugene, OR): 2.0 mM solu-
tion in DMSO; store at −20°C (see Note 6).
11. Vectashield gel mounting medium (Vector Laboratories, Inc.,
Burlingame, CA).
12. Confocal scanning microscope equipped with an argon laser
source for fluorescence measurements (for example, Leica
TCS SP5, Mannheim, Germany).
13. FACS tubes.
14. Fluorescent-activated cell sorting FACS equipment and
appropriate software (for example: FACSCanto (BD, San
Jose, CA) using the FACSDiva Software (BD, Milan, Italy)).

1. Lyophilized Ab42, (trifluoroacetate salt, Sigma) and Ab42
amine-reactive succinimidyl esters of carboxyfluorescein
(Ab42-FAM) (see Note 6) (AnaSpec, San Jose, CA) is dis-
solved and incubated in hexafluoro-2-isopropanol (HFIP)
(Sigma) to a final 1.0 mM concentration for 1.0 h at room
temperature to allow complete peptide monomerization. The
Ab42 and Ab42-FAM solutions are dried under N 2 and stored
in aliquots at −20°C.
2. Aliquots of Ab42 are dissolved in DMSO to a final concen-
tration of 5.0 mM and incubated to a concentration of
100 µM in ice-cold F-12 Ham nutrient medium (Sigma) for
24 h at 4°C.
3. Ab42-FAM aggregates are obtained using a mixture of Ab42-
FAM peptide with 2 molar equivalents of unlabeled Ab42
peptide (at ratio of 1:2) (see Note 7).

2.3.1. Preparation
of Amyloid b -Derived
Diffusible Ligands