Biology Reference
In-Depth Information
1.2 Materials and Methods
1.2.1
Isolation and Culture of AFSCs
AFSCs were isolated from amniotic fluid samples by centrifugation and
resuspended in
-MEM growth medium supplemented with 20% FCS, 1% penicil-
lin/streptomycin, 1%
L
-glutamine, and 5 ng/ml fibroblast growth factor (FGF).
Cells were incubated in a Petri dish in a humidified 38.5
C/5% CO
2
incubator. At
80% confluency, cells were lifted with 0.05% trypsin EDTA and counted using a
Burker chamber.
a
1.2.2 Nucleofection
10
5
) were resuspended in 100
Cells (6
l of Human MSC Nucleofector Solution
m
(Amaxa Biosciences) with 2.5
g pAcGFP-N1 vector (Clontech) and nucleofected
with the U-23 or C-17 programs of a Nucleofector II device. These programs are
specific for human MSCs. Immediately after nucleofection, cells were plated into
90-mm dishes and incubated for 48 h. After the incubation, gentamycin (400
m
g/ml)
was added to growth medium to select for the cells with internalized and integrated
plasmid. The selection was performed for 2 weeks.
m
1.2.3 GFP Expression Assessment
Samples of AFSCs nucleofected with either of the two programs were used to
assess fluorescence as determined by GFP expression. Cells were stained with
propidium iodide (PI), resuspended in 500-
l of phosphate-buffered solution
(PBS), and analyzed on a flow cytometer Coulter Epics XL (Beckman Coulter).
The fluorescence signals of GFP and PI were measured in channels FL1 and FL2,
respectively, with a logarithmic scale.
m
1.2.4 Assessment of Stemness Markers
Immunocytochemistry and RT-PCR were used to assess stemness of nucleofected
cells. Cells (2
10
4
) were fixed in 4% paraformaldehyde/PBS for 15 min at room
temperature and were analyzed by immunocytochemistry for the expression of
SOX2, NANOG, and TERT pluripotency markers. For RT-PCR analysis, total
RNA was extracted from 1
10
6
AFSCs using TRI Reagent (Sigma). Reverse
