Cruzfluor 594

After cholinergic neuronal induction of hDPSCs-cryo, immunostaining for cholinergic neuron-specific marker proteins was performed using previously described protocols [37 (link)]. Briefly, DF-chN were fixed with 3.7% formaldehyde for 40 min, permeabilized with 0.2% Triton X-100 supplemented with 1% BSA, and thereafter blocked with 1% BSA (in DPBS) at 20 °C for 1 h. Cells were then incubated with primary antibody, diluted 1:100, choline acetyltransferase (ChAT; ab68779, Abcam, Cambridge, UK), homeobox HB9 (motor neuron and pancreas homeobox 1, MNX1) (HB9; sc-22542, Santa Cruz), and insulin gene enhancer protein ISL-1 (ISL1; sc101072, Santa Cruz) at 20 °C for 1 h. Following incubation with primary antibodies, cells were washed with DPBS and incubated with 1:200 CruzFluor^TM 594 or CruzFluor^TM 488 conjugated donkey anti-rabbit or donkey anti-goat or donkey anti-mouse IgG secondary antibodies (Santa Cruz) for 1 h. For nuclear staining, cells were treated with 1 µg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 min at 20 °C. Finally, cells were observed under a fluorescence microscope (Nikon Eclipse Ti-U, Nikon Instrument).

HEK293T cells were seeded at 1.7 × 10⁵ cells/cm² within 10 mL of DMEM with 10% FBS and 1% penicillin-streptomycin in a T-75 flask. The next day, 5 µL of pcDNA3.1-hACE2 plasmid (Addgene) was mixed with 0.1 mL of PEI for 30 min and then added to the cells for transfection. The culture medium was replaced with a fresh one. After 48 h, 500 µg/mL G418 disulfate (Sigma-Aldrich) was used for selection for up to four weeks. The stable HEK293T cell line expressing hACE2 (HEK293T-ACE2) on the surface of the outer cell membrane was confirmed by incubating HEK293T-ACE2 cells with anti-ACE2 mAb for 1 h at room temperature. Following three gentle PBS washes, cells were treated for 25 min at room temperature with a fluorescent secondary antibody: anti-mouse IgGκ binding protein conjugated with red fluorescent dye CruzFluor^TM 594 (Santa Cruz Biotechnology). A Leica DMi8 microscope equipped with a Leica EC3 camera (Leica Microsystems, Wetzlar, Germany) was used to image the cells. Western blot analysis was performed in HEK293T-ACE2 cells by using RIPA lysis buffer (Thermo Fisher Scientific). Briefly, after performing SDS-PAGE for the cell lysate, the gel was transferred onto the nitrocellulose membrane. ACE2 mAb and HRP-conjugated mouse IgG secondary antibodies were used for Western blot detection.