PROTOCOL FOR TUMORSPHERE ASSAYS WITH CAF AND RMF CM 1. Thaw CM in waterbath. Thaw only what will be used for the experiment- do not freeze/thaw and reuse CM. 2. Supplement CM with 5% serum, antibiotics and L-glutamine (if CM is PRF) so cells will survive a week of culture. Volume of CM needed for duration of expt: ________________ 5% CS or CD-FBS (specify) of __________ total CM volume is ________ mL CS needed per CM tube. 1X AB/AM for ____________ total CM volume is __________ mL 100X AB/AM stock needed per CM tube. 2 mM L-glutamine for ___________ total CM volume is ________ mL 200 mM Lglutamine stock needed per CM tube. 3. Once CM is supplemented, add appropriate volume to nonadherent culture vessel to precoat the wells or plates before adding cell suspension. For one well of a 6 well plate, add 2 mLs per well. For a p60, add 2.5 or 3 mLs per plate, depending on the total volume of CM available. 4. Trypsinize MCF7 cells to a single cell suspension. 5. Count MCF7s with a hemocytometer. If you see large clumps of cells on hemocytometer and not single cells, go back and pipet up and down to achieve a single cell suspension. Use this technique to ensure a seeding density of single cells; if this is not done properly it will severely impact your experiment! 6. Resuspend MCF7s in a volume that brings the concentration to 106 cells/ mL. For resuspension, use the media that was used to collect the CM necessary for this experiment. Media used for fibroblast conditioning: ___________________________ # cells/mL suspension: __________________ # cells within suspension: ___________________ volume needed for 106 cells/mL: __________________ 7. Seed 30,000 cells per well of a 6 well plate or 40,000 cells per p60. Seed evenly and with a single cell suspension. Type of culture vessel: _____________________ Seeding density: ___________________ Volume/ well needed for seeding density above: ________________ 8. Feed the cultures on day 3 with a fresh 2 mLs of supplemented CM. Just add this directly to the wells or plate. 9. Take photos of the cultures and measure the size of the spheres before the conclusion of the expt to ensure that they are within the size constraints of the multisizer. 10. At conclusion of experiment, harvest the cells for sphere quantification following Patty’s instructions.