By Schulz L., Kapitany A.
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The Par polarity complex regulates Rap1- and chemokine-induced T cell polarization. J Cell Biol, 176(6), 863–875 (2007). Osmani N, Vitale N, Borg JP, EtienneManneville S. Scrib controls Cdc42 localization and activity to promote cell polarization during astrocyte migration. Curr Biol, 16(24), 2395–2405 (2006). Nobes CD, Hall A. Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia. Cell, 81(1), 53–62 (1995).
This is simple and does not require time-lapse microscopy, but it does not provide any information on individual cell speed or persistence. 2. 1). Perform each condition in triplicate. After 3 h (see Note 7), remove growth medium and fix cells with 4% formaldehyde or ice-cold methanol, depending on the antibody you will be using (step 3). 2% Triton X-100 in PBS, 5 min. Block cells with 1% BSA and stain with desired antibody according to the manufacturer’s instructions. Co-stain with fluorescent phalloidin and DAPI.
Excess water is drained and the coverslips are incubated in an oven at 150°C overnight for sterilisation before use. The coverslips are stored at 4°C in sterile conditions until required for use. Different colony-forming cell types can be used in the HGFinduced cell scatter assay. These include MDCK cells (12), MCF-7 breast carcinoma cells (16), and HT29 colon carcinoma cells (17). The protocol can be adapted and optimised by, for example, changing the cell density or HGF concentration as required.