Fluorescence Assays to Study Cell Adhesion and Migration In Vitro - Methods in Molecular Biology: Extracellular Matrix Protocols - page 313

15. Microplate fluorometer capable of reading independently from top and bottom of

the plate (SPECTRAFluor Plus; TECAN Group).

2.2. FATIMA
1.

Cells to be assayed for their capability to transmigrate/invade a 2- and 3-dimen-
sional molecular substrate ( see Note 14 ).

2. Bicarbonate buffer as used for CAFCA

3. Fluorescent cell labeling reagents : A) Labelling dye solution : 0.25 M sucrose

(0.85 g per 10 mL apirogen H 2 O), store at 4 ° C. The solution should be sterilized

using a 0.2- m filter. B) Stock dye solution : Fast DiI™ and Fast DiO™ (Molecu-

lar Probes, Inc.) are prepared in ethanol at 1-5 mg/mL and stored at -20

µ

C. Cen-
trifugation of the concentrated solution before addition to the cells is
recommended to remove the undissolved dye crystal ( see Note 15 ).

°

4. Transwells (Costar, Cambridge, MA), Unicell 24 plates, FATIMA plates (Whatman

Polyfiltronics) carrying membranes with different pore sizes ( see Note 16 ).

5. Purified extracellular matrix molecules. Matrigel should be handled as indicated

in the instructions provided by the supplier ( see Note 17 ).

6. PBS and EDTA as used for CAFCA.

7. Serum-free DMEM or RPMI as used for CAFCA.

8. 2% bovine serum albumin (BSA; stock solution). 2.0 g of BSA in 100 mL

DMEM/RPMI. Sterilize using a 0.2-

µ m filters and store in aliquots at -20 ° C.

9. Working assay solution: RPMI or DMEM containing 0.1-0.5% BSA.

10. Conditioned medium from fibroblastic cells to use as generic chemoattracting

agent. We normally use the conditioned medium from the NIH 3T3 fibro-
blasts grown in DMEM containing 10% FCS. When cells are at 70-80%
confluency, remove the supernatant gently, wash with PBS and replace medium
with 10 mL serum-free DMEM. After 24 h, collect the supernatant, centrifuge,
sterilize using a 0.2- µ m filter, add BSA (0.1-0.5 % final concentration) and store

at -20 ° C until use.

11. Cotton swabs to remove nontransmigrated cells from the upper side of the porous

membrane of the transmigration unit (not necessary when using FATIMA plates).

12. Microplate fluorometer, such as the SPECTRAFluor Plus, capable of reading

independently from top and bottom of the plate.

3. Methods
3.1. CAFCA
3.1.1. Procedure for Cell-Substratum Adhesion
1. Prepare the “coating solution” composed of the ECM molecule(s) of interest dis-

solved at 0.01-100

µ g/mL (total protein concentration) in the bicarbonate buffer

and aliquot 50 µ L in each well of the bottom CAFCA miniplate ( see Fig. 1 and

see Note 1 ).

2. Incubate the bottom CAFCA miniplates at 4 ° C for 8-16 h ( see Note 2).

3. Dissolve the BSA at 1% (w/v) in the bicarbonate buffer and heat up the solution

to 56 ° C for 15 min to denatured the protein ( see Note 3 ).