Live/Dead cell Staining Kit II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Prepare a staining solution of 2 µM Calcein-AM/4 µM EthD-III by adding 5 µl of 4 mM Calcein-AM and 20 µl of 2 mM EthD-III to 10 ml of PBS or other serum-free buffer or medium.	Incubate the cells for 15 minutes at room temperature

Upstream tips
Prepare a staining solution of 2 µM Calcein-AM/4 µM EthD-III by adding 5 µl of 4 mM Calcein-AM and 20 µl of 2 mM EthD-III to 10 ml of PBS or other serum-free buffer or medium.
Protocol tips
Incubate the cells for 15 minutes at room temperature

Publication protocol

NIH/3T3 fibroblast (Cell bank of National Institute of Health Sciences (Japan)) was a gift from Professor Naoki Tanaka (Kyoto Institute of Technology). The cells were cultured using Dulbecco’s modified Eagles’s medium (DMEM) supplemented with calf serum (volume fraction of 10%) and penicillin/ treptomycin (volume fraction of 1%). Cells were incubated at 37 °C under 5% CO2. Cells grown to 80% confluence were passaged by trypsinization, diluted, and inoculated into a fresh tissue culture dish. Fibroblasts were seeded at a density of ca. 1000 cells cm −2 onto RGDS-ELP or RGDS-deg-ELP-self-assembled PSt-dishes in the absence of serum at 37 °C under 5% CO2. For comparison, RGDS-free oligo(ELP) was also used. After incubating for 3, 6 and 24 h, the number of attached cells per surface area and the degree of spreading of the cells (the number of spreading cell/the number of attached cell × 100) were determined by optical phase contrast microscopy (CKX41, Olympus Co., Tokyo, Japan). Four independent cell adhesion experiments were performed, and the average values were used. Errors include all result of 4 independent experiments. Cell sheet recovery experiment was carried out as follows. The NIH/3T3 cells (ca. 2000 cells·cm −2
) were cultured on RGDS-ELP-self-assembled PSt dish in the presence of serum at 37 °C for nine days. After these cells became confluent, the cells were washed by phosphate buffer solution (PBS) (37 °C) and
then incubated in serum-free DMEM at 20 °C for 5 h. Cell viability assays for surface-attached cells were conducted using the live/dead assay kit (Promokine
TM Promocell Co., Heidelberg, Germany). NIH/3T3 cells (ca. 1.0 × 10 4 −2 cells·cm) were cultured at 37 °C on various surfaces for 24 h. Then, the cells were stained with calcein (2 μM) and ethidium D (EthD) (4μM) for 15 min. Fluorescence images were obtained on a CKX41-FL (Olympus Co.) equipped with a digital camera system (QIClick, Nippon Roper Co., Tokyo, Japan). The live cells correspond to green cells and the red ones to dead cells.

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