Cells in the upper compartment were removed, leaving only the cells on the underside of the filter- these represented those cells who had successfully invaded across the collagen-coated filter.
After incubation for 36 h at 37 °C, the invasive cells on the underside of the membrane and the noninvasive cells on the top of the membrane were harvested aseptically and expanded for the next round of selection.
These cells are gradually displaced to the underside of the mantle by more recent cells in the groove, and switch to secreting the mineralized material of the shell valves.
Invasion of cells to the underside of the Matrigel-coated membrane was detected by staining the cells with Mayer's hematoxylin solution and visualizing the cells under a microscope.
Simultaneous depletion of TSG101 and MMP-9 reduced numbers of migrated cells on the underside of the filter to the levels seen in the cells treated with MMP-9 siRNA duplexes alone (Fig. 5c, d).
Uncircumcised men are thought to be more susceptible because the underside of the foreskin is rich in Langerhans cells, sentinel cells of the immune system, which attach easily to the human immunodeficiency virus, which causes AIDS.
Cells present on the underside of the transwell (i.e., migrated cells) were counted by microscopy.
The numbers of invading cells on the underside of the membrane were significantly reduced both in SW480 cells (Fig. 4a, P < 0.01) and SW620 cells (Fig. 4B, P < 0.01) when transfected with miR-217 mimics compared to those transfected with NC mimics.
The total number of invading cells was determined by counting the cells on the underside of the insert from triplicate wells (six fields per insert) at 20× magnification.
To evaluate the amount of cells that had invaded through each transwell, excess of media was discarded and the transwells were washed once with PBS and then placed in trypsin solution (0.025%) to release the invaded cells underside of the transwells and in the bottom chamber.
Depletion of TSG101 using TSG#1 or TSG#2 siRNA duplexes led to increased numbers of migrated cells on the underside of the filter (Fig. 1d, e), suggesting that TSG101 is involved in cell invasion of HT1080 cells.
