284 Chapter 11 8. Flow cytometry analyses of dissociated cardiac spheroids 1. Collect the spheroids in a 15 mL tube, and centrifuge for 3 min at 70xg. 2. Aspirate the supernatant and add 1 ml PBS. 3. Centrifuge for 3 min at 200xg for 5 min. 4. Aspirate the supernatant and dissociate the CSs by adding 1 mL of TrypLE Select Enzyme solution. 5. Incubate the tube at 37 °C for 15 min. 6. Using a 5 mL pipette, mechanically dissociate the cells by flushing with 2 mL RPMI-1640 so that single cells can be seen when observed under a microscope. 7. Centrifuge for 3 min at 200xg for 5 min. 8. Aspirate the supernatant and add fix the CMs with 200 μl 4% PFA. 9. Incubate for 10 minutes at room temperature. 10. Centrifuge for 3 min at 200xg for 5 min. 11. Aspirate the supernatant and add 1 ml PBS. NOTE: Pause Point: The fixed hiPSC-CMs can be stored at 4°C for up to 4 weeks. 12. Transfer the cell suspension to a FACS tube. 13. Centrifuge for 3 min at 200xg for 5 min. 14. Aspirate the supernatant and resuspend 1 × 105 cells in 50 μl of permeabilization buffer. 15. Incubate the cells for 30 min at 4 °C. 16. For immunofluorescence flow cytometry analysis: 16.1 Resuspend in 50 μl of flow cytometry buffer containing the α-actinin antibody (1:300 dilution) and in another FACS tube resuspend 1 × 105 cells in 50 μl of flow cytometry buffer with the respective isotype control (e.g., FITC mouse IgM, κ isotype (1:200 dilution)) and 1 × 105 cells in 50 μl of flow cytometry buffer for negative control. 16.2 Incubate the cells for 30 min at 4°C. 16.3 Wash the cells with 2.5 ml of flow cytometry buffer and centrifuge at 200xg for 5 min at 4°C; discard the supernatant and repeat the wash two more times. 16.4 Resuspend in 50 μl of flow cytometry buffer containing the secondary-antibody Goat-anti-mouse (1:300 dilution). NOTE: Place the tube in the dark since the secondary-antibody solution is lightsensitive. 17. For viability check with propidium iodide (PI), add 1:1000 PI and incubate for 15 min. NOTE: Place the tube in the dark since the PI solution is light-sensitive. 18. Analyze the cells with a flow cytometer, adjusting the gates according to the standard gating strategy as shown in supplementary figure 1.
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