Remove 6 well plate from incubator Aliquot 1.8 ml of Poly-NIPAM to the chilled 15 mL tube using a positive displacement pipette Add appropriate amount of cells (50 ul-using calculation above) to gel and stir with tip (do not resuspend with this tip as the gel is very viscous) Add 150 uLs of cell/ Poly-NIPAM suspension to one well in the 6 well plate using positive displacement pipette Hold pipette as vertical as possible; try not to add any bubbles to the gel by pipetting at a steady pace both when taking the suspension from the 15 mL tube and when placing the gel in the well Once all the gels have been pipetted into wells, place plate in incubator for 5 minutes When ready, add 3 mL of media to each well and return to incubator to allow spheroid formation
Remove 6 well plate from incubator Aliquot 1.8 ml of Poly-NIPAM to the chilled 15 mL tube using a positive displacement pipette Add appropriate amount of cells (50 ul-using calculation above) to gel and stir with tip (do not resuspend with this tip as the gel is very viscous) Add 150 uLs of cell/ Poly-NIPAM suspension to one well in the 6 well plate using positive displacement pipette Hold pipette as vertical as possible; try not to add any bubbles to the gel by pipetting at a steady pace both when taking the suspension from the 15 mL tube and when placing the gel in the well Once all the gels have been pipetted into wells, place plate in incubator for 5 minutes When ready, add 3 mL of media to each well and return to incubator to allow spheroid formation
Spheroid-Culture_Protocol.docx