Fluorescence Assays to Study Cell Adhesion and Migration In Vitro - Methods in Molecular Biology: Extracellular Matrix Protocols - page 314

4. Remove the coating solution from the wells, wash them at least 2-3 times with

bicarbonate buffer, and fill them with 200 L of the BSA blocking solution (or

analogous solution). Incubate at room temperature for at least 2 h ( see Note 4 ).

µ

5. Fluorescent cell labeling: Suspension-growing cells: Rinse the cells once and

resuspend them in 300 µ L of RPMI with 15% FCS. Add calcein AM (at final

concentration of 1-10 µ M ; the recommended calcein AM-cell ratio is 2 µ M /10 6

cells) and incubate the cells at 37 C for 10-20 minutes to allow the calcein AM

to be metabolized. The incubation time with calcein AM may in some cases need
to be extended, especially when working with ex vivo cells. The optimal labeling
is achieved when the cell pellet attains a yellowish color.
Anchorage-dependent cells: Remove the culturing medium and extensively
rinse the plates with PBS, followed by incubation with 2 mL PBS (for a 35-mm
plate) containing 5 m M EDTA. Incubate the cells in the presence of the EDTA
for up to 15 min (most cells should detach from the culture dish within 5 min, but
we have found that some cells may require somewhat longer incubation times).
Collect the cell suspension, wash the cells by centrifugation to remove all EDTA,
and resuspend them in 300

°

L of DMEM (or another preferred medium). Cells
can then be labeled with calcein AM as indicated for suspension-growing cells
( see Note 5 ).

µ

6. Remove the blocking agent from the wells and wash them at least twice with 200 µ L

of the cell-adhesion medium containing PVP. Fill then the wells with 200 L of

the cell adhesion medium in which 2% (v/v) India ink has been added ( Fig. 1 )
( see Note 6 ).

µ

7. Collect the fluorescently labeled cells by centrifugation, rinse them twice with

cell-adhesion medium containing PVP and resuspend them in the chosen cell-
adhesion medium at the appropriate concentration. Aliquot 50 µ L of the cell sus-

pension in each well ( see Note 7 ).

8. Place the bottom CAFCA miniplates in the apposited bottom CAFCA black

holder ( Fig. 2 ) and centrifuge them at 142 g for 5 min, followed by incubation
37 C for 20 min ( Fig. 1 ) ( see Note 8 ).

9. Fill the wells of the top CAFCA miniplate with the same PVP- and India
ink-containing medium as used for the bottom CAFCA miniplates, such as to
assure to form a bulging meniscus ( Fig. 1 ). Place a bottom CAFCA miniplate
into the apposited light-metal assembly device with the handle-rod ( see Fig. 2 ).
Fill also the wells of the bottom CAFCA miniplates with a similar excess of
liquid ( see Note 9 ).

°

10. Take a top CAFCA miniplate and reverse it upside down in the air ( Fig. 1 ) by

holding it from the ends (not from the middle as it tends to bend). Avoid touching
the double-sided tape, especially when wearing gloves (e.g., such as those that
may be worn when handling infectious cells and/or solutions). Because of the
high liquid surface tension in such a narrow well, the liquid will not fall out from
the upside-down oriented well. Bring the top CAFCA miniplate in this reversed
orientation in proximity of the corresponding bottom CAFCA miniplate, making
sure to align perfectly the “top” and “bottom” wells. First, gently bring in con-