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1. Splitting SBNET Spheroids
NOTE: This is done for expansion and for sharing with other researchers.
- Use a P1000 pipette to mechanically break the ECM and aspirate the ECM with SBNET spheroids to a sterile 1.5 mL tube.
- Centrifuge at 1,000 x g at 4 °C, remove all the supernatant and place the tube on ice.
- Add 2-4x the volume of the new ECM to the pellet. Mix the new ECM with the old ECM and SBNET spheroids by pipetting up and down 10x, avoiding introducing air bubbles.
- Transfer 5-20 µL of the ECM and SBNET spheroids mixture to a new plate and allow ECM to solidify.
- Cover with new SBNET medium and transfer to the incubator. The recovery rate is approximately 95% to 100%.
- For shipping SBNET spheroids to another lab, transfer SBNET spheroids with new ECM to a T25 flask and allow ECM to solidify.
- Fill the T25 flask with SBNET spheroid medium, screw on the cap tightly, and prepare the shipment package.
- Upon receiving an SBNET spheroid culture, remove the culture medium and perform steps 1.1 to 1.5 to put SBNET spheroids back in culture.