Brdu detection kit

pPICs and human myoblasts were plated in 24-well plates at a density of 5 × 10³ per well and were serum starved for 6 h in 0% serum DMEM/F12 medium. To investigate the effect of pPIC conditioned media on proliferation, wells were either supplemented with standard GM, unconditioned medium (UM), pPIC conditioned medium (CM), or heat-inactivated conditioned medium (ICM). Each well was supplemented with BrdU (1 μg/ml) every 8 h, fixed in 4% paraformaldehyde after 24 h, and BrdU incorporation was then assessed using the BrdU detection kit (Roche) (n = 3/condition).
In order to investigate the effect of growth factors on pPIC proliferation, wells were serum starved for 6 h and then switched to basal media (control), or supplemented with IGF-1 (100 ng/ml; Peprotech), HGF (100 ng/ml; Peprotech), NRG-1 (100 ng/ml; R&D Systems, Minneapolis, Minnesota), or TGF-β1 (5 ng/ml; Peprotech). Each well was supplemented with BrdU (1 μg/ml) every 8 h, fixed after 24 h, and BrdU incorporation was assessed using the BrdU detection kit (Roche) (n = 3/growth factor). Nuclei were counterstained with DAPI. Cells were evaluated using an ApoTome fluorescent microscope (Carl Zeiss). The percentage of BrdU^pos cells relative to the total number of cells was determined by counting 5 random fields at ×20 magnification for each dish, and then expressed as fold change over unsupplemented control.

To analyze the proliferation rate of mouse hepatocytes, we used primary hepatocyte culture conditions as previously described.^{41 (link)} The percentage of cells undergoing DNA synthesis was estimated by counting the number of BrdU-labeled cells using the BrdU detection kit (Roche Diagnostics, Meylan, France). Cells were incubated with BrdU-labeling medium for 5 h. The medium was then removed, and cells were fixed in ethanol/acetic acid (7 : 3) and stained according to the manufacturer's instructions. Ten separate fields per plate were counted.
Cultured primary hepatocytes were fixed by 4% PFA, stained by rabbit anti-Ki67 (Thermo Fisher Scientific) and mouse anti-PCNA (Thermo Fisher Scientific) primary antibody, followed by donkey anti-rabbit Dylight488-conjugated and goat anti-mouse Dylight549-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) at 37 °C for half an hour in dark. Nuclei were stained with Dapi-Fluoromount-G (Sourthernbiotech). The positive nuclei and DAPI in a field were counted, five separate fields per plate were counted.