N5413

Passage (P) 2 Hip-NSCs were treated with the thymidine analog BrdU (Sigma-Aldrich, St Louis, MO, USA) at 10 µmol/l upon reaching 50% confluency. A total of 48 h later, the neurospheres derived from the Hip-NSCs were plated on polylysine-treated sterile glass coverslips and incubated at 37°C in 5% CO₂ for 2–4 h. Following thorough rinsing with phosphate-buffered saline (PBS), the neurospheres were treated with 2 M hydrochloric acid for 1 h at 37°C and boric acid buffer (pH 8.5) for 10 min at room temperature (RT). BrdU-incorporated cells were visualized by immunostaining. Briefly, cells were incubated with blocking solution 0.3% Triton X-100 containing 1% normal donkey serum (Jackson ImmunoResearch Labortories, Inc., West Grove, PA, USA) for 30 min. Cells were then incubated with a primary antibody mixture of mouse anti-BrdU (1:200; ab8152; Abcam, Cambridge, MA, USA) and rabbit anti-nestin (1:100; N5413; Sigma-Aldrich) for 24 h at 4°C, followed by the secondary antibodies (Alexa Fluor 488-conjugated donkey anti-mouse IgG; 1:1,000; R37114; Alexa Fluor 594-conjugated donkey anti-rabbit IgG; 1:1,000; R37119; both Thermo Fisher Scientific, Inc.) for 4 h at RT. Finally, the immunostained Hip-NSCs were mounted onto glass slides using VECTASHIELD^® antifade fluorescence mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA), and observed under a fluorescence microscope.

Following 9 days of differentiation in vitro, the cultures were trypsinized into single cells and washed once with FACS buffer [PBS plus 1% fetal bovine serum (FBS)]. The cells were fixed in Cytofix™ Fixation Buffer (BD Biosciences, Franklin Lakes, NJ, USA) for 15 min at room temperature. Following 3 washes with FACS buffer, permeabilization was performed using Phosflow™ Perm/Wash Buffer I (BD Biosciences) for 10 min at 4°C. The cells were then incubated with primary antibody against Nestin (1:200; N5413; Sigma-Aldrich, St. Louis, MO, USA) diluted in Phosflow™ Perm/Wash Buffer I for 15 min at 37°C. The cells were washed with FACS buffer 3 times, followed by incubation with secondary antibody (1:500; A11008; Life Technologies, Eugene, OR, USA) in FACS buffer for 15 min at 37°C. The cells were washed and resuspended in PBS. Analyses were performed using an Accuri C6 flow cytometer (BD Biosciences) and FlowJo software.

Anti-nestin and oct4 immunostaining were employed to
identify the stemness of AD-MSCs. Briefly, the cells were
fixed by 4% paraformaldehyde solution for 20 minutes
at room temperature and washed three times in PBS.
[For the BrdU-labeling assay, the cells were fixed using
70% ethanol for 10 minutes, and they were exposed to
2 M of HCl for 1 hour at 37°C. Subsequently, the cells
were incubated in 0.1 M of borate buffer (pH=8.5) for 10
minutes]. Next, the cells were permeabilized with 0.3%
Triton X-100 in PBS for 15 minutes and 10% goat serum
for 15 minutes at room temperature and were allowed to
incubate overnight at 4°C with primary antibodies such
as anti-BrdU primary antibody (1:500; B 2531; Sigma,
USA), anti-nestin (1:500; N5413; Sigma, USA) and
anti-Oct4 [(1:100); ab18976; rabbit anti Oct4 antibody,
Abcam, Cambridge, UK]. Next, the cells were washed
twice in PBS and stained with anti-mouse secondary
antibody Rhodamine-conjugated (1:100; Millipore,
USA, AP124R), fluorescein isothiocyanate (FITC)-
conjugated secondary anti-mouse (1:100) for nestin and
FITC-conjugated secondary anti-rabbit (1:100) for Oct4
at 37°C for 1 hour. Cells were examined by fluorescent
microscope (E600 Eclipse; Nikon, Netherlands) equipped
with a digital camera (DXM 1200; Nikon Digital).