Labtek 2 cc2 chamber slide

To check the mitochondrial integrity, PAMs were seeded at 2 × 10⁵ cells per well on a LabTek II CC2 chamber slide (154941, Thermo Fisher). The cells were infected with the Sk02 virus at an MOI of 1 for 7 h or they were stimulated with 200 ng/mL LPS for 16 h. After fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in Dulbecco’s phosphate-buffered saline (DPBS) for 5 min, the cells were incubated with 5% BSA in DPBS for 1 h. The cells were probed with mouse monoclonal anti-TOM20 antibody (sc-17764, Santa Cruz, Dallas, TX, USA) and goat polyclonal anti-IAV antibody (AB1074, EMD Millipore, Etobicoke, ON, Canada) overnight at 4 °C, and the secondary antibodies (Alexa Fluor 488 donkey polyclonal anti-mouse IgG_H+L (A21202; Invitrogen, Mississauga, ON, Canada) and Alexa Fluor 633 donkey polyclonal anti-goat IgG_H+L (A21082; Invitrogen)) for 1 h at room temperature. Counterstaining was done with 4′,6-diamidino-2-phenylindole (DAPI) (D1306, Invitrogen) for 5 min and the coverslips were mounted with ProLong Diamond Antifade Mountant (P36961, Invitrogen) overnight at room temperature. Images were obtained by using a confocal laser scanning microscope (TCS SP8, Leica, Concord, ON, Canada).

On the ninth day of differentiation, aggregated SHED-β cells were transferred on the Lab-Tek II CC2 Chamber Slide (Thermo Scientific Inc., New York, NY, USA) and incubated for 24 h to allow proper attachment on the surface. The cells were fixed with CytoCell Fixative solution (Biocare Medical, Concord, CA, USA) for 20 min, and incubated in blocking solution (CAS-BLOCK; Invitrogen, Halethorpe, MD, USA) for 15 minutes at room temperature. SHED-β cells were stained with anti-Insulin (GenScript, Piscataway, NJ, USA) antibody at room temperature for 2 hours, washed three times with PBS, and then incubated with Alexa Fluor 594-conjugated secondary antibody (Invitrogen, Eugene, OR, USA) for 1 h. After being washed with PBS, the slide was mounted in Vectashield mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) (Vector Laboratories, Burlingame, CA, USA). Fluorescent images of the samples were obtained using a Zeiss LSM530 META Confocal Microscope (Carl Zeiss, Thornwood, NY, USA).

Cells were grown on Labtek II-CC2 Chamber slides (Nunc), fixed with 4% paraformaldehyde for 5 minutes, washed with PBS and blocking buffer (PBS with 5% BSA and 0.1% Triton X- 100), and then incubated overnight at 4°C in primary antibodies against YAP1, AR and CK5. Secondary antibodies were Donkey anti-mouse, -rabbit or -goat coupled to Alexa-350, -488 or -647 (Invitrogen). Cell nuclei were visualized with DAPI (Sigma).