1C: T: 400 mM urea is greater in osmotic strength than a physiological solution (i.e.hyperosmotic). However, a RBC membrane is freely permeable to urea, therefore has much lower tonicity (hypotonic). In 400 mM urea, the cell would swell and lyse as if suspended in a solution of deionized water.
2B: The rate of cell shrinkage and crenation depends on membrane permeability to water; i.e., the faster water can exit the cell, the faster the cell can shrink in response to hypertonic solution. The greater the degree of hypertonicity of the solution (concentration of membrane impermeant solutes), the greater the osmotic force available to drive water out of the cells, causing shrinkage and crenation (force drives flow).
For the cell motility assay, trypsinized cells were resuspended in serum-free RPMI 1640 medium supplemented with 0.1% bovine serum albumin (BSA). Cell suspensions were seeded in the upper chamber of a transwell insert with an 8 μm diameter (Corning, Tewksbury, MA, USA), and complete medium was added to the lower chamber. After incubation for 24 h, cells on top of the membrane were displaced with a cotton swab, and cells on the bottom of the membrane were fixed in methanol and stained with crystal violet. Cells in three fields were counted per membrane. For the invasion assay, the transwell was coated with 200 μL Matrigel at 200 μg/mL and pre-incubated with RPMI1640 medium. Cells were seeded into the upper chamber of the transwell (20,000 cells/insert) and RPMI 1640/BSA was added to the lower chamber. After 24 h of incubation at 37C, cells were fixed in methanol and stained with crystal violet or DAPI. Cells that invaded through the pores to the lower surface of the filter were counted under a microscope. Three invasion chambers were used per condition, and the total number of cells from the three filters was averaged. For the wound-healing assay, 1 106 cells were seeded on 6-cm plates coated with 10 μg/mL type I collagen. Cells were incubated for 24 h, the monolayer was disrupted with a cell scraper (1.2 mm width), and photographs were taken at 0 and 24 h with a phase-contrast microscope. Experiments were carried out in triplicate, and four fields of each condition were recorded.
Human umbilical vein endothelial cells (HUVECs) were obtained from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. Cells were co-cultured with breast cancer cells using a double-chamber method in 24-well plates as described . MDA-MB-231 cells (1 104 cells) were seeded into transwell chambers consisting of polycarbonate membranes with 0.45-μm pores and allowed to adhere overnight. The chambers were then placed into the HUVEC culture and were co-cultured for 4 to 8 days. Medium of HUVECs was removed and the collagen gel prepared as previously described  was applied to the cells. Fresh DMEM containing 3% FBS was then added to the collagen gel and cells were incubated at 37C for 24 h. The reorganization of the subconfluent monolayer HUVECs was monitored and photographed with a phase contrast microscope (Nikon). 153554b96e