Dispase assay (cell-cell adhesion assay)
Prepared by: Nichole Lobdell (2008)
Modified from: Calautti et al, JCB, 1998/ Green et al, PNAS, 2005/ Green et al, JCB,
2002
Cell lines tested: pBabe, MCF10A, NeuN, NeuT, CA1a, CA1d, p120-kd (+ control?)
1.
Seed duplicate 60mm dishes of cells and grow to ~confluence (can wait one day past
confluence to make sure that everyone is adhering).
2.
Wash the plate twice in PBS.
3.
Incubate in 2ml Dispase for 20-40 minutes at 34C. Start checking the cells at 20
minutes and recheck every 5 minutes after that for adhesion. Make sure to do the
same thing for every comparison sample.
4.
When the cells pull off the plate in a sheet, add 5 ml PBS to the plate and then take
all 7 ml and spin in the centrifuge to pellet the cells.
5.
Carefully remove the liquid without disrupting the pellet.
6.
Add back 1 ml PBS and pipette the pellet 15, 30, or 45 times ( depends on the cell
line of interest) to disrupt them. Be sure not to bubble the cells when pipetting and
keep it constant between samples. Then I count 15 ul on a hemacytometer.
7.
Count single cells or fragments. I prefer fragments because I do see a difference in
the amount of fragments ( this is how the Green lab does it ). The third option is that
you can respin the sample after pipetting and only get the single cells in suspension
to count them. I never got the correct speed to get the single cells suspension as the
Calautti paper referenced. Your choice!
8.
Respin the pellet, pull off the PBS, and add 1ml trypsin to the pellet.
9.
Incubate at 37C for 7 minutes.
10. Pipette this preparation for the same number of times ( I find the MCF cells still need
this extra pipette step because they don’t just fall apart in the trypsin ).
11. Count 15 ul of this sample.
12. Dividing the number of cells disassociated by the dispase by the total number of
cells in the trypsin, will give a percent disassociation. The higher the number, the
less adhesive your cells.
NOTE: The dispase I am using was referenced by one of the papers: Roche Dispase II:
#10295825001