Dear Darshini!

Please You see in this protocol:

Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations

Article Generation of human induced pluripotent stem cell-derived ca...

Characterization of hiPSC-CMs by immunofluorescent staining

Dissociated single cells (1×106) were plated on Matrigel-coated round cover slips in 12-well plates and incubated for two days in a humidified incubator at 37°C and CO2 level of 5% in 1 mL/well RPMI 1640 supplemented with 1× B27 without insulin and 50 μg/mL ascorbate. Alternatively, immunocytochemistry was performed directly with hiPSC-CMs that were generated on Matrigel-coated square cover slips in six well plates. The wells were washed with 1× DPBS-/-, fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature, and permeabilized with 0.5% Triton X-100 in DPBS-/- for 15 minutes. Intact cardiac clusters were fixed and permeabilized with 100% methanol for 15 minutes. Unspecific antibody binding sites were blocked by incubation in 1 mL per well (or for clusters, in 1 mL per 15 ml tube) of 3% bovine serum albumin (BSA) (Sigma-Aldrich, # A2153) in DPBS-/- for one hour at room temperature. For preparation of cryoslices, cardiac clusters were embedded in Tissue-Tek (Sakura, # 1227900003) and stored frozen at -80°C, and cryo-sliced at 8 µm thickness. Cells or cardiac cluster samples were incubated overnight at 4°C in 3% BSA containing 1:200 dilution of troponin T (TNNT2) rabbit polyclonal IgG (Abcam, # ab45932) and 1:200 dilution of α-actinin (ACTN2) mouse monoclonal IgG1 (Sigma-Aldrich, # A7811). The next day, cells were washed three times with DBPS-/- and incubated for 60 minutes at room temperature in the dark and 3% BSA containing Hoechst 33342 and Alexa Fluor 555 goat α-rabbit IgG (Thermo Fisher Scientific, # A21430) and Alexa Fluor 647 goat α-mouse IgG (Thermo Fisher Scientific, # A21236), both at a concentration of 1 µg/ml. Following the final washing steps, cells were mounted with 5 µL SlowFadeTM Diamond Antifade Mountant (Thermo Fisher Scientific, # S36972) and examined by confocal fluorescent microscopy (SP8, Leica).

Interested

Hi Alexandr,

Thank you very much for this, i will try this!