Ultra low adhesion 24 well dishes

Primary tumorsphere formation was assessed in cells grown under anchorage-independent conditions in methylcellulose. BT549 (15,000) or LM2-4 cells (10,000) were cultured in 0.9 mL of 1% methylcellulose/complete DMEM medium in ultra-low adhesion 24-well dishes (Corning, Corning, NY, USA) and cells cultured for 10−12 days. Primary tumorspheres were assessed by counting colonies consisting of at least 6 cells from 4 fields per well with a 10× objective. We measured self-renewal by collecting primary tumorspheres by dilution in at least 3 volumes of PBS, dissociating them with trypsin for approximately 10 min, and re-seeding in 1% methylcellulose before evaluating secondary colonies after 10 days. For treatment with rhTGFBI (R&D Systems), a single dose of 500 ng/mL was added only once, when initially embedding the cells, and compared against cells receiving the same volume of vehicle (PBS).

Primary tumorsphere formation in methylcellulose was assessed in cells grown under anchorage-independent conditions. LM2-4 cells (10,000) were cultured in 0.9 mL of 1% methylcellulose/complete DMEM medium in ultra-low adhesion 24-well dishes (Corning, Corning, NY, USA) and cells cultured for 8–10 days. Cells were treated with vehicle (DMSO) or the indicated doses of SP600125, JNK–IN–8, or Reversine (all from Selleckchem) concurrent with embedding in methylcellulose. Primary tumorspheres were assessed by counting colonies consisting of at least 6 cells from 4 fields per well with a 10x objective.