Cellstain hoechst 33342 solution

Cells were washed with PBS and then immersed in 4% paraformaldehyde PBS and fixed for 10 min. Permeabilization Buffer (10×) from eBioscience (Vienna, Austria) was used instead of 0.1% Triton X-100 in PBS. Liquid 1% FBS added to PBS was used as an antibody diluent.
Immunofluorescence staining was performed using specific antibodies for Troponin T, Cardiac Isoform Ab-1 (Clone 13-11; Thermo Fisher Scientific K.K.), Anti-β-actin (C4; Santa Cruz Biotechnology) and Anti-mouse IgG, HRP-linked Antibody (Cell Signaling Technology). Cellstain^®-Hoechst 33342 solution (DOJINDO LABORATORIES, Kumamoto, Japan) was used for nuclear staining of the cells. Images were recorded using a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan). Since SeV is carried on the SeV vector, SeV-positive cells can be positively identified with a GFP light source without the need for fluorescently labelled antibodies. Tra-1-60 was then purchased from R&D Systems, Inc. and used for live-staining. After fluorescent staining, cells were detached with trypsin according to the usual cell detachment method and then counted after making cell suspensions with StemFit AK03 containing ROCK inhibitor (10 μM).

Cells were labeled with 10 μM BrdU (B1575, Tokyo Chemical Industry) for 1 hr before harvest. Trypsinized cells (1 × 10⁶ cells) were washed two times with ice-cold PBS and fixed in 1 mL of 70% ethanol for no less than 16 hr at 4°C. After washing two times with PBS, fixed cells were denatured by incubating in 500 μL of 2.0 M HCl for 30 min at 25°C. Denatured cells were washed two times with PBS and once with 1 mL of sodium borate buffer, pH 8.5. The cells were permeabilized in 500 μL of PBS containing 0.1% Triton X-100 (PBSTx) for 10 min at 25°C. The permeabilized cells were incubated for 1 hr at 25°C in 500 μL of PBS containing 1% bovine serum albumin (BSA), Alexa Fluor 488-conjugated anti-BrdU antibody (1:100, 3D4, BioLegend), and Cellstain Hoechst 33342 solution (1:100, H342, Dojindo). The cells were then washed once with 1 mL of PBS containing 1% BSA (PBS/BSA) and resuspended in 500 μL of the same buffer. The stained samples were analyzed by BD LSRFortessa X-20 and FlowJo v10.6 (Becton Dickinson).