90 Chapter 4 • CM differentiation medium + 8 μM CHIR: In a sterile environment, add 0.02% CHIR (1:5000 dilution of the 40 mM CHIR stock) into CM differentiation medium. Note: After preparation, do not store this medium to avoid repeated freeze-thaw cycles of CHIR. • Wnt-C59 (20 mM): In a sterile environment, add 1302.2 µL DMSO to 10 mg Wnt-C59. Note: Prepare aliquots of 50 µL format and store at −80°C for up to 2 years. Avoid refreezing of the aliquots. • CM differentiation medium + 2 μM Wnt-C59: In a sterile environment, add 0,01% Wnt-C59 (1:10.000 dilution of the 20 mM Wnt-C59 stock) into CM differentiation medium. Note: After preparation, do not store this medium to avoid repeated freeze-thaw cycles of Wnt-C59. •CM culture medium (CM): In a sterile environment mix RPMI 1640, 2% B-27 supplement (1:50) and 1% penicillin/streptomycin (1:100). • CM purification medium (PM): In a sterile environment mix RPMI 1640 no glucose, 2% B-27 supplement (1:50) and 1% penicillin/streptomycin (1:100). • CM replating medium (RM): In a sterile environment, add 0.1% Y-27632 (1:1000 dilution of the 10mM stock) and 10% KO serum replacement (1:10) into CM culture medium. Note: After preparation, all cardiac culture mediums can be stored at 4°C for up to 1 month. CM expansion medium (EM) (with 2 µM CHIR): In a sterile environment, add 0.005% CHIR (1:20.000 dilution of the 40 mM CHIR stock) into CM culture medium. Note: After preparation, we do not recommend storing this medium. • CM splitting medium (SM) (with 2 µM CHIR): In a sterile environment, add 0.005% CHIR (1:20.000 dilution of the 40 mM CHIR stock), 0.1% Y-27632 (1:1000 dilution of the 10mM stock), and 10% KO serum replacement (1:10) replacement into CM culture medium. Note: After preparation, we do not recommend storing this medium. • CM thawing medium (TM): In a sterile environment, add 1% Revitacell (1:100) and 10% KO serum replacement (1:10) into CM culture medium. Protect medium from light. CRITICAL: Revitacell is light-sensitive. Protect Revitacell from light. Note: After preparation, this medium can be stored at 4°C for up to 1 week. Note: We recommend to prepare aliquots of 100-500 µL Revitcell and store at −20°C for up to 1 year. • Permeabilization and blocking buffer: This buffer contains PBS, 5% BSA and 0,3% Triton-X-100. • Flow cytometry buffer: This buffer contains 50 ml of PBS and 1% BSA and 0.3% Triton-X-100.
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