then pipet up and down several times to recover all the Triton
X-100 sticking to the tip walls.
18. Dextran sulfate can be difficult to get into solution. To dissolve
it, vortex the solution and mix on a rocker overnight.
19. MCF10A doubling time is about 24 h.
20. At D0, wait for all cells to be attached before adding TGF-β to
the medium. We recommend to wait 24 h when using the
MCF10A cell line.
21. Epithelial cells are closely packed and adopt polyhedral shapes
(see Fig. 1a) while mesenchymal cells are elongated and make
few cell-to-cell contacts (see Fig. 1b).
22. Wait minimum 48 h after trypsin treatment to harvest the cells.
23. For MCF10A, we use a cell counter with a threshold set-up at
10 μm.
24. To ensure band sharpness, the height of the stacking gel should
be at least twice the height of the sample in the well.
25. Isopropanol both prevents contact with oxygen that inhibits
acrylamide polymerization and enables to level the
resolving gel.
26. The rest of the mix is used to follow the polymerization: if
polymerization has occurred in the container, gel will be polymerized in the cassette.
27. Time of polymerization depends on several parameters (thickness of the gel, TEMED and APS concentrations and freshness,
acrylamide concentration). If polymerization hasn’t occurred
after 45 min to 1 h, it has a great chance to never polymerize.
In that case, start all over again, with fresh APS and TEMED.
28. Be prompt to use or store the gel once polymerization has
occurred as the gel can start drying out with time.
29. If not to be used immediately, wrap gel, with comb still
inserted, in a well-humidified paper towel overlaid with a plastic wrap. Store flat at 4
C for 3 days maximum as pH and ionic
strength gradually disappear during storage.
30. If pellet remains undissolved, sonicate your samples. We use a
Bioruptor Plus with the following parameters: 3 cycles, 30 s
time ON, and 30 s time OFF. We do as many sonicating cycles
as needed (number of cycles may vary upon sample). Each
sample should be homogeneous.
31. Centrifugating helps recovering any condensates and will pellet
insoluble residues.
32. Make sure to pull out slowly and vertically the comb in order to
avoid bending the wells, introducing bubbles and gel pieces
into the wells. If this occurs, try to take out bubbles and gel
pieces by pipetting up and down buffer into the well with a
378
Noe ´ mie Kempf et al.
X-100 sticking to the tip walls.
18. Dextran sulfate can be difficult to get into solution. To dissolve
it, vortex the solution and mix on a rocker overnight.
19. MCF10A doubling time is about 24 h.
20. At D0, wait for all cells to be attached before adding TGF-β to
the medium. We recommend to wait 24 h when using the
MCF10A cell line.
21. Epithelial cells are closely packed and adopt polyhedral shapes
(see Fig. 1a) while mesenchymal cells are elongated and make
few cell-to-cell contacts (see Fig. 1b).
22. Wait minimum 48 h after trypsin treatment to harvest the cells.
23. For MCF10A, we use a cell counter with a threshold set-up at
10 μm.
24. To ensure band sharpness, the height of the stacking gel should
be at least twice the height of the sample in the well.
25. Isopropanol both prevents contact with oxygen that inhibits
acrylamide polymerization and enables to level the
resolving gel.
26. The rest of the mix is used to follow the polymerization: if
polymerization has occurred in the container, gel will be polymerized in the cassette.
27. Time of polymerization depends on several parameters (thickness of the gel, TEMED and APS concentrations and freshness,
acrylamide concentration). If polymerization hasn’t occurred
after 45 min to 1 h, it has a great chance to never polymerize.
In that case, start all over again, with fresh APS and TEMED.
28. Be prompt to use or store the gel once polymerization has
occurred as the gel can start drying out with time.
29. If not to be used immediately, wrap gel, with comb still
inserted, in a well-humidified paper towel overlaid with a plastic wrap. Store flat at 4
C for 3 days maximum as pH and ionic
strength gradually disappear during storage.
30. If pellet remains undissolved, sonicate your samples. We use a
Bioruptor Plus with the following parameters: 3 cycles, 30 s
time ON, and 30 s time OFF. We do as many sonicating cycles
as needed (number of cycles may vary upon sample). Each
sample should be homogeneous.
31. Centrifugating helps recovering any condensates and will pellet
insoluble residues.
32. Make sure to pull out slowly and vertically the comb in order to
avoid bending the wells, introducing bubbles and gel pieces
into the wells. If this occurs, try to take out bubbles and gel
pieces by pipetting up and down buffer into the well with a
378
Noe ´ mie Kempf et al.