92 Chapter 4 Table 2. key resources table REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-α-actinin, primary mouse monoclonal antibody Merck, A7811 Anti-Ki67, primary rabbit polyclonal antibody Abcam AB833 Propidium Iodide (PI) Cell Signaling 4087S Goat Anti-mouse Alexa Fluor 488 Invitrogen A11029 Goat Anti-rabbit Alexa Fluor 568 Invitrogen A11011 Chemicals, peptides, and recombinant proteins B-27 supplement minus insulin (B27-insulin) Thermo fisher A1895601 B-27 supplement plus insulin (B27+insulin) Thermo fisher 17504-044 Bovine Serum Albumin Fraction V Roche 10735086001 CHIR-99021 Selleckchem S2924 EDTA Thermo fisher 15575020 Essential 8 (E8) medium Thermo fisher A1517001 Paraformaldehyde solution 4% in 1x PBS, pH 7.0 – 7.6 Santa Cruz SC281692 PBS, pH 7.4 Thermo fisher 10010072 KnockOut™ (KO) Serum Replacement Thermo fisher 10828 Matrigel, growth Factor Reduced, Basement Membrane Matrix Corning 356230 Penicillin/streptomycin Thermo fisher 15140 Triton X-100 Merck X100-1L TrypLE™ Select Enzyme (10X) Thermo fisher A12177 Trypan Blue Solution, 0.4% Thermo fisher 15250061 RevitaCell Thermo fisher A2644501 ROCK inhibitor Y-27632 Biorbyt orb60104 RPMI 1640 medium Thermo fisher 11875 RPMI 1640 medium no glucose Thermo fisher 11879020 STEMdiff™ Cardiomyocyte Freezing Medium Stemcell 05030 Wnt-C59 Tocris 5148 Experimental Models: Cell Lines Human induced pluripotent stem cell lines (Stanford Cardiovascular Institute (S-CVI) Biobank) CVI-111 (control 1) CVI-114 (control 2) CVI-273 (control 3) STEP-BY-STEP METHOD DETAILS hiPSC culturing Timing: 30 min 1. When the hiPSCs are 80–90% confluent (usually about 3 to 4 days after passaging) aspirate the old medium, then add 1 ml room temperature 0.5 mM EDTA to each well. At this point, 2.5 to 3 million cells should be present in each well of the 6-well culture plate. 2. Incubate the plate at room temperature and wait for 3-5 minutes. 3. Aspirate the EDTA and flush the hiPSC with a 1 ml tip filled with 1 ml E8 + 5 μM Y27632, dispense the medium over the surface of the plate well until all the cells are detached and gently mix 2-5 times using the 1 ml tip.
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