Calcein am pi double staining kit

Pyrene (purity > 98%) was obtained from TCI (Tokyo Chemical Industry, Tokyo, Japan). Polymers of branched polyethylenimine (BPEI) were obtained from Aladdin (Shanghai, China). A Cell Counting Kit-8 (CCK-8) and Calcein-AM/PI Double Staining Kit were purchased from dojindo (Dojindo Laboratories, Kumamoto, Japan). Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated fetal bovine serum (FBS), Doxorubicin (DOX), and trypsin were purchased from BI (Shanghai, China). Furthermore, 4,6-diamidino-2-phenylindole (DAPI) and DII were purchased from beyotime (Shanghai, China).

HaCaT cells were pretreated without or with SIP (5, 10, 20, 50 μg/mL) for 24 h, followed by exposure to UVB (200 mJ/cm²) in PBS. The cells were then washed with PBS and incubated for 6 h in DMEM. After that, the cells were stained with a Calcein-AM/PI double staining kit (Dojindo, Tokyo, Japan) and then examined under a fluorescence microscope.

hCMs were dissociated with 0.25% trypsin/EDTA (25200072, Gibco) and then seeded into 384‐well plates at the density of 8 × 10³ cells per well. 24 h after seeding, cells were exposed to the drugs at the indicated concentrations for 6 days. Culture media were fully changed every 3 days with new drug supplementation. After drug treatment, cells were stained with calcein‐AM/PI/Hoechst for 20 min using calcein‐AM/PI double staining kit (C542, DOJINDO). Images were captured by the Operetta CLS High‐Content Analysis System (PerkinElmer). Number of live cells (calcein‐AM+/PI‐) and dead cells (PI+) was quantified by using the Harmony 4.9 software (PerkinElmer). Number of live cells was used to calculate the “relative cell viability” as follows
$$\begin{array}{c}\hfill \mathrm{relarive}\mathrm{cell}\mathrm{viability}=\frac{\mathrm{number}\mathrm{of}\mathrm{live}\mathrm{cells}\mathrm{in}\mathrm{each}\mathrm{treatment}}{\mathrm{number}\mathrm{of}\mathrm{live}\mathrm{cells}\mathrm{in}\mathrm{DMSO}\mathrm{control}}\times 100\%\end{array}$$

Cell viability was analyzed by Calcein‐AM/PI Double Staining Kit (Dojindo, Japan). Briefly, each scaffold in 24‐well plates was soaked in 1 mL mixture solution (AM:PI:PBS = 2:1:1000) in dark conditions for 20 min. The live cells with green fluorescence excited by 488 nm laser and dead cells with red fluorescence excited by 556 nm laser were observed by a fluorescence microscope (DMi8 S, Leica, Germany).

The staining operation was performed in accordance with the instructions of the Calcein-AM/PI double staining kit (DOJINDO, C542, China). In a 24-well plate, after the scaffold was wetted with culture medium, the cells were seeded at 2 × 10⁴ on the scaffold. After the cells adhesion, more culture medium was added, and live/dead staining was performed after 3 and 7 days. On a clean bench, the medium was discarded, the scaffold was washed twice with PBS and put into a 15 ml centrifuge tube; 10 µL of light-sensitive Calcein-AM stock solution was added. Additionally, 15 µL of PI stock solution was added to 5 ml of PBS solution to make a homogeneous working solution. Under dark conditions, 1 ml of PBS solution and 500 µL of dye solution were added to each well, incubated for 15 min in the incubator, and images were collected under a fluorescence confocal microscope (Leica, TCS. SP8, Germany).

Cell viability was assessed using the Calcein-AM/PI double-staining kit (Dojindo). In brief, cells were incubated with 2 μM calcein-AM, along with 3 μM PI in DPBS for 20 min at 37 °C, followed by washing twice with DPBS after incubation. Fluorescence images were taken with a confocal microscope (FV3000, Olympus). Live and dead cells were observed as green and red fluorescent signals, respectively. According to the microscopic scanned picture, the cell viability was quantified using Fiji ImageJ software.

Human PDLSCs (8 × 10⁴ cells/scaffold) were seeded on aligned and random scaffolds (15 mm × 7.5 mm × 0.5 mm, L × W × H) in a nonadherent 24-well plate and cultured in the growth medium. On day 7, samples were incubated with a working solution of calcein-AM/PI Double Staining Kit (Dojindo, C542) for 20 min in a 37 °C incubator. The cells were then observed under a fluorescence microscope.