Cell observer living cells

NHMs were inoculated on cell climbing sheets at a density of 10⁴ cells at 37°C with 5% CO₂ for 24 h. After being washed three times with 0.1M PBS buffer solution (Solarbio, Beijing, China, P1010‐2L), cells were fixed with 95% ethanol at room temperature for 15 min and dried at room temperature. The cells were then blocked with bovine serum for 30 min at 37°C and washed three times with 0.1 m PBS buffer solution again. Following incubation with the prime antibody (mouse monoclonal antihuman OPN3, 1:50; MDL, MD6636‐100) at 4°C overnight, the cells were washed with 0.1 m PBS buffer solution for 5 min three times and covered with goat anti‐mouse IgG FITC‐labeled fluorescent antibody (1:50; MDL, MD6640‐100) for 1 h at 37°C, finally stained by 40,6‐diamidino‐2‐phenylindole (DAPI) (Gibco, D21490). The expression of OPN3 in NHMs was visualized under the confocal microscope (ZEISS, German). NHMs were also double labeled with mouse monoclonal antihuman OPN3 (1:50; MDL, MD6636‐100) and rabbit monoclonal antihuman melan‐A (1:50; Bioss, Beijing, China, bs‐7362R). The immunohistochemistry protocol used was described above, and cells were visualized under Cell Observer‐Living Cells (Zeiss).

The TSiN‐H2B‐RFP lentiviral construct was a kind gift from Dr. P. J. Galardy (Mayo Clinic). Lentivirus was prepared by transfecting HEK293T cells with the TSiN‐H2B‐RFP lentiviral plasmid, a psPAX2 packaging plasmid, and a pMD2.G envelope plasmid. A172 cells were infected with lentivirus encoding H2B‐RFP in the presence of 8 μg/mL polybrene. Time‐lapse imaging was then performed using a Cell Observer (Cell Observer Living Cells, Carl Zeiss) equipped with a camera. Frames were recorded every 5 minutes. Cell morphology was visualized under a phase‐contrast microscope, and red fluorescence was detected as described previously.27

The third passage NHMs (60% confluency) were transfected for 48 h with a final siRNA concentration of 30 nm as described above. Cells were washed once with 0.1 m PBS buffer solution and incubated with 1 mL M254 medium containing 2.5 μm Fluo‐3/AM (MULTISCIENCES (LIANKE) BIOTECH, Hangzhou, China, F1243) for 30 min at 37°C in the darkroom. Fluorescent images of Fluo‐3/AM‐loaded cells were acquired using Cell Observer‐Living Cells (Zeiss, German). Controls included no siRNA transfection and negative control siRNA. In addition, cells were also harvested and incubated with 1 mL M254 medium containing 2.5 μm Fluo‐3/AM for 30 min at 37°C in the darkroom. Fluo‐3/AM‐loaded NHMs were centrifuged at 1000 g for 5 min at room temperature, washed one time with 0.1 m PBS buffer solution and resuspended with 0.5 mL 0.1 m PBS buffer solution. Then, the concentration of intracellular free calcium ion was measured with flow cytometric assay (BD Biosciences, San Jose, CA). The excitation source for Fluo‐3/AM was a 488‐nm air‐cooled argon laser and a 525‐nm band‐pass filter.

TSiN-H2B-RFP lentiviral construct was kindly gifted by Dr. P. J. Galardy (Mayo Clinic). Lentivirus was prepared by transfection of HEK293T cells with TSiN-H2B-RFP lentiviral plasmid, psPAX2 packaging plasmid, and pMD2.G envelope plasmid. A549 cells were infected with lentivirus encoding H2B-RFP in the presence of 8 μg/ml polybrene. Time-lapse imaging was acquired using a Cell Observer (Cell Observer Living Cells, Carl Zeiss) equipped with a camera and Axiovision (Carl Zeiss). Frames were recorded every 5 min. Cell morphology was visualized on a phase-contrast microscope, and RFP was detected by fluorescence [68 (link)].