285 Generation, high throughput screening, and biobanking of human iPSC-derived cardiac spheroids 11 9. Immunofluorescence staining of whole 3D spheroids NOTE: during the procedure, all pipet tips and tubes can be coated with 1% BSA-PBS to prevent the spheroids from sticking to plastics. 1. Collect the spheroids (approx. 20-50 spheroids per antibody combination) into a 15 mL coated tube by using a 5 mL pipet. NOTE: Be careful not to damage the spheroids. 2. Centrifuge for 3 min at 70xg and aspirate the supernatant. 3. Carefully resuspend the organoids in 1 mL of ice-cold PFA using a coated 1 mL tip. 4. Fix at 4 °C for 45 min. Gently resuspend the spheroids halfway through the fixation time using a coated 1 mL tip to ensure even fixation among all spheroids. 5. Add 10 mL of ice-cold PBT to the tube, gently mix by inverting the tube, incubate for 10 min and spin down at 70 x g, both at 4 °C. NOTE: From this step onwards coating of tips is generally not needed as most organoid types do not stick to the tip after fixation. However, some organoids may require coated plastics even after fixation. 6. Block the spheroids by resuspending the pellet in ice-cold OWB (at least 200 μL of OWB per well) and transfer the spheroids to a 24-well suspension plate. NOTE: Organoids from one large pellet can be split over multiple wells to perform different stainings. Use approx. 20-50 spheroids per antibody combination. 7. Incubate at 4 °C for at least 15 min. 8. Pipette 200 μL of OWB in an empty well to serve as a reference well . NOTE: The immunolabeling can also be performed in 48- or 96-well plates to reduce antibody usage. However, the user should be aware that both staining and washing performance could be reduced due to the smaller volume. 9. Allow the spheroids to settle at the bottom of the plate, by leaving the plate at 45° for 5 minutes. 10. Remove OWB leaving the organoids in 200 μL of OWB (use the reference well to estimate 200 μL). 11. Add 200 μL of OWB with primary antibodies 2x concentrated (e.g., Alpha-actinin [1:200] and Troponin T [1:200] for results in Figures 1C and 4D and incubate overnight at 4 °C while mildly rocking/shaking (40 rpm on horizontal shaker). 12. The next day, add 1 mL of OWB. 13. Allow the spheroids to settle at the bottom of the plate by leaving the plate at 45° for 5 minutes. 14. Remove OWB leaving 200 μL in the plate. Add 1 mL of OWB and wash for 2 h with mild rocking/shaking. 15. Repeat steps 13 and 14 for two more times. 16. Allow the organoids to settle at the bottom of the plate by leaving the plate at 45° for 5 minutes.
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