Cell adhesion molecules (CAMs) are a class of membrane surface glycoproteins. They mediate the contact and binding between cells and between cells and extracellular matrix and regulate cell functions in a ligand-receptor specific binding manner. They play an important biological role in many physiological and pathological processes such as embryonic development and differentiation, maintenance of normal tissue structure, inflammatory response, immune response, coagulation and thrombosis, wound repair, tumor infiltration and metastasis.
Experimental steps:
1. Pre-coat 96-well plates with 10ug/ml Fn, 70ul/well, overnight at 4℃, and wash 3 times with PBS
2. Block with 1% BSA at 37℃ for 1h, wash 3 times with PBS
3. Cultivate the cells to be tested to the logarithmic growth phase, digest the cells, suspend the cells with serum-free medium, count them with a hemocytometer, and adjust the concentration to 5*105/ml
4. Inoculate them in 96-well plates pre-coated with Fn, 5000 cells per well, and set 3 duplicate wells for each group. After incubation at 37 °C for 1 h, wash away non-adherent cells with PBS
5. Fix with 5% glutaraldehyde at 4 °C for 0.5 h, wash 3 times with PBS; stain with 0.1% crystal violet, let stand at room temperature for 0.5 h, wash 3 times with PBS
6. Add 100 μl of 10% acetic acid to each well, and detect the absorbance value at 595 nm with an enzyme reader after 5-10 min to indicate the number of adherent cells
7. Alternatively, add 100 ul of methanol to each well and fix for 15 min
8. Add 100 ul of Giemsa stain to each well, stain for 15 min, and wash away the stain with PBS
9. Randomly select 5 random fields of view under an inverted microscope to count the number of adherent cells and take photos, and statistically calculate the results
Warm reminder:
If you use an enzyme reader to count, follow steps 1-6; if you need photos, follow steps 1-4, 7-9.