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For cell cycle analysis of cells embedded in soft agar, cells (10 5 ) were suspended in 1 mL of 0.33% noble agar, seeded in the bottom of 15-mL polypropylene tubes, cooled for 5 min on ice, and then kept at 37 C in a humidified incubator in the presence of 5% CO 2 for 24 h72 h