In situ death detection kit

Cells were rinsed twice in PBS and fixed in 4% paraformaldehyde for 20 min. After washing, cells were incubated with Hoechst 33258 staining solution (Beyotime Institute of Biotechnology) at room temperature for 5 min. The apoptotic cells were then observed under a fluorescent microscope (Zeiss Inc., Oberkochen, Germany). DNA fragmentation was visualized by the TUNEL method using the TUNEL Apo-Green Detection Kit (Biotool, Houston, TX, USA). Cells were fixed and permeabilized. After being washed, cells were incubated with TdT terminal transferase and FITC-12-dUTP, and then counterstained with DAPI. For heart sections, apoptotic cardiomyocytes were examined using the In Situ Death Detection Kit (Roche, Branchburg, NJ, USA), with myocytes counterstained by α-actinin antibody (Sigma-Aldrich, St. Louis, MO, USA) and observed using a Zeiss fluorescent microscope. TUNEL-positive nuclei were quantitated by counting 1000 random cardiomyocytes and calculated using the Image J software (NIH, Bethesda, MD, USA).

In order to investigate the cell viability, M17 cells
were seeded at a density of 5×10³ in 96-well plates and
incubated overnight while keeping the temperature
constant at 37˚C. The addition of the exosomes derived
from control-miR and hAD-MSC-miR-124-Cy3 to
M17 cells was carried out after 24 hours. The flow
cytometry analysis was performed to detect the delivery
of exosomes containing miR-124. Upon the assurance of
the delivery of miR-124 to the M17 cells, the viability
of M17 cells transfected with miR-124 and control miR
was examined by the MTT assay after 24, 48 and 72
hours. The rate of apoptosis in M17 cells transfected with
miR-124 and control miR was assayed using terminal
deoxynucleotidyltransferase dUTP nick-end labeling
(TUNEL) assay performed by the In Situ Death Detection kit (Roche Diagnostics, Indianapolis, IN) according to the
manufacturer’s instructions. The cultured M17 NB cells
with the secreted exosomes from hAD-MSCs-Con-miR
(M17-hAD-MSCs-Con-miR) were treated with derived
exosomes from hAD-MSCs-miR-124 (M17-hAD-MSCsmiR-
124). The cultured M17 NB cells were also directly
transfected with miR-124 (M17-miR-124) and its control
(M17-con-mir).
In this study, the data were analyzed by the Statistical
Package for Social Sciences version 11 software (SPSS,
IBM, USA).

TUNEL assays were performed on 10-μm frozen, apical cross-sections of hearts. Cryotome-cut frozen sections were first dried for 10 min at room temperature and fixed for 10 min in 4% PFA. Sections were subsequently used for TUNEL staining (In situ Death Detection Kit, Cat. 1684795, Roche) following manufacturer's instructions. After TUNEL, primary antibody anti-α-Actinin (Sigma-Aldrich, A7811) was applied overnight at 4°C with a concentration of 1:800. Secondary antibody incubation was performed at room temperature for 1 h, followed by 5 min Hoechst incubation and mounting with Fluoromount (Southern Biotech). Images were taken by a blinded investigator using the Olympus BX63 and cellSens and analyzed in ImageJ.

Apoptotic nuclei were labelled using the terminal deoxynucleotidyl transferase uridine nick end labelling (TUNEL) technique with an In Situ Death Detection Kit, POD (Roche, Nutley, NJ, USA). A light haematoxylin stain was used for tissue orientation following 3, 3′-diaminobenzidine (Sigma)/H₂O₂ rinses. The number of apoptotic cells stained by TUNEL assay in the hippocampal CA1 region was counted as following: An observer blinded to the study group counted the total number of apoptotic nuclei per high-power field (HPF) in six non-overlapping regions per section (×400).

Apoptotic cells in frozen liver-tissue samples (−80°C; O.C.T.-included; 14-months-old mice, n = 3) were detected by TUNEL staining, using the In Situ Death Detection Kit (Roche, 1 684 795). Images were acquired with a Leica DM2500 confocal microscope.