Live dead cell staining kit 2

Live/Dead cell staining was performed following the manufacturer's instructions (Live/Dead Cell Staining Kit II, PromoCell GmbH, Germany).

On each scaffold, 100 µL of the medium were pipetted with 25,000 cells/100 µL of MG-63. The well plates were then incubated for 2 h at 37 °C and at a CO₂ saturation of 5% in an incubator. After two hours, 1 mL of the specific cell medium described previously was added to each well before incubating the well plates in the incubator for 3, 7, and 10 days. The staining solution was prepared by adding 2 mL DPBS (art. no. 14190-094, Gibco, Grand Island, NE, USA) to a Falcon tube (Greiner Bio-One International GmbH, Kremsmünster, Austria) and 4 µL ethidium homodimer III (Eth D-III) solution (together with the calcein part of the Live/Dead Cell Staining Kit II (PromoCell, Heidelberg, Germany)), according to the manufacturer’s protocol (PromoCell). Then, 1 µL of calcein dye was added after mixing the staining solution. All steps were performed in the dark to avoid photobleaching of the staining solution and samples. To eliminate serum esterase activity, all samples at a specific time point had the medium removed and the cells washed. Staining was then performed according to a previously published protocol [34 (link)]. The evaluation was performed using an Olympus fluorescence microscope (BX51, Olympus, Osaka, Japan) at 5 different positions on the samples, at 5× and 10× magnification.

Acetaminophen (APAP), collagen, penicillin/streptomycin, Williams’ Medium E, hydrocortisone hemisuccinate, insulin, dimethyl sulfoxide (DMSO), DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride), Mowiol and 6-carboxyfluorescein diacetate were purchased from Sigma-Aldrich (Steinheim, Germany); fetal bovine serum and glutamine were purchased from Biochrom (Berlin, Germany). For immunofluorescence staining, the following antibodies were used: anti-ZO1 mouse IgG (BD Biosciences, Heidelberg, Germany), anti CYP2E1 rabbit and anti CYP3A4 rabbit IgG (Bioss Antibodies, Woburn, MA, USA), AlexaFluor594 labeled goat anti-mouse and AlexaFluor488 labeled goat anti-rabbit antibody (Invitrogen, Darmstadt, Germany). A live/dead cell staining kit II was purchased from Promocell.

To evaluate cytotoxicity, MC3T3-E1 cells were seeded at a density of 5 × 10⁴ cells/scaffold onto CNTps and IP-CHAs placed in 48-well plates, and cultured for one and five days (n = 7 for each group). After staining by H33342, cells were observed by an IX71 inverted microscope at 400× magnification.
To compensate the limited field depth of 3D-culturing, multiple Z levels were integrated by the Zerene Stacker software (Zerene Systems, Richland, WA, USA). Consequently, cells in a single 400× field were counted.
Live/dead cell staining was done on the first and third day of incubation to visualize the cell viability. The cells were incubated with 0.1 µM Calcein-AM and 2 µM Ethidium homodimer-III (EthD-III) at 37 °C for 30 min (Live/Dead Cell Staining Kit II; PromoCell GmbH, Heidelberg, Germany). Stained cells were viewed using fluorescence microscopy (IX71, Olympus, Tokyo, Japan). Viable cells were stained with Calcein AM and fluoresced green, while dead cells were stained with EthD-III and fluoresced red.

RAW 264.7 and NIH/3T3 cells were plated in a 24-well plate at a density of 1 × 10⁵ and 5 × 10⁴ cells/well, respectively, and incubated overnight. Subsequently, the cells were cultured in a treatment medium containing carboxylated PRXs (10 mM threaded β-CD) for 24 h at 37°C. Next, they were stained using a Live/Dead Cell Staining Kit II (PromoCell, Heidelberg, Germany) for 30 min at 37°C; in this procedure, calcein-AM (2 μM) and ethidium homodimer III (EthD III; 4 μM) stained live and dead cells, respectively. The phase contrast and fluorescent images of the stained cells were acquired using an IX-71 (Olympus) equipped with a DP-80 dual CCD microscope camera.

UHP treated cells (4 × 10⁴ cells) were washed with PBS, and then the cells were suspended into live/dead solution which was prepared by following the provided manual (Live/Dead Cell Staining Kit II; PromoCell GmbH, Germany). The cells were incubated at 37°C for 1 hour. After the incubation, images were obtained using an Olympus FluoView confocal laser scanning microscope (Olympus, Tokyo, Japan).