281 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 7. Prepare Pluronic solution 1. Mix pluronic powder F-127 (20% final concentration) and PBS. Filter using a 0.22 μm vacuum-driven filter and store at 4 °C. Equilibrate to RT before use. 8. Prepare calcium dye medium 1. Mix the Pluronic F-127 solution (final concentration of 0.04%) and the Cal-520 AM (1:1000) in fluor bright medium: In a 50 mL conical tube, add 10 uL of Cal520, 20 μL of Pluronic solution. Mix until fully dissolved. Keep the solution in the dark. 2. Preparation of Buffers Prepare Permeabilization and blocking buffer: This buffer contains PBS, 5% BSA, and 0,3% Triton-X-100. Prepare the Flow cytometry buffer: This buffer contains 50 ml of PBS and 1% BSA and 0.3% Triton-X-100. Prepare the Flow cytometry washing buffer: This buffer contains 50 ml of PBS and 1% BSA. Prepare Spheroid washing buffer: This buffer contains 1 mL Triton-X-100, 2 mL of 10% (w/v) SDS, and 2g BSA in 1L PBS. NOTE: OWB can be stored at 4 °C for up to 2 weeks. Prepare the FUnGI solution: This solution contains 50% (v/v) glycerol, 9.4% (v/v) dH2O, 10.6 mM tris base, 1.1 mM EDTA, 2.5M fructose, and 2.5 M urea. Note: FUnGI can be stored at 4 °C in the dark. FUnGI should not be heated as fructose caramelizes at higher temperatures. Preparation time = 1 day. Prepare PBT buffer. This buffer contains PBS/Tween-20 (0.1% v/v)): For 1L of PBS add 1mL Tween-20. 3. Preparation of Small Molecules NOTE: Reconstitute all small molecules in DMSO unless otherwise stated. 1. Reconstitute 10 mM aliquots of 50 µL each of Thiazovivin (ROCK inhibitor) and store at -20 °C. 2. Prepare 2.5 mM aliquots of 10 µL each of Cal-520 AM and store them at -20 °C.
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