FA disassembly/reassembly assays were performed as previously described (Ezratty et al., 2005 (link), 2009 (link); Nader et al., 2016 (link)). Briefly, keratinocytes were grown on fibronectin-coated coverslips until confluent and then serum starved for 18–24 h in serum-free low calcium E media. The keratinocytes were then treated with 10 μM NZ (Sigma-Aldrich M1404) in serum free low calcium E media for 3–4 h, washed with PBS, and placed in fresh serum-free low calcium E media. At the desired time points, the cells were then either fixed for 10 min with 4% PFA for subsequent IF or scraped off the cell coverslip with a cell scraper and incubated with antibodies for subsequent flow cytometry.
Protocol detail
FA Disassembly/Reassembly Assays in Keratinocytes
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Antibodies
Assays
Calcium
Cell
Fibronectin
Flow cytometry
Keratinocytes
Low serum media
Serum
Serum free media
Corresponding Organization :
Other organizations : Columbia University
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Variable analysis
independent variables
- Treatment with 10 μM NZ (Sigma-Aldrich M1404) in serum free low calcium E media for 3–4 h
- Disassembly/reassembly of focal adhesions (FA) as measured by immunofluorescence (IF) or flow cytometry
- Keratinocytes grown on fibronectin-coated coverslips until confluent
- Keratinocytes serum starved for 18–24 h in serum-free low calcium E media
- Cells washed with PBS and placed in fresh serum-free low calcium E media after treatment
- Positive control: Not explicitly mentioned
- Negative control: Not explicitly mentioned
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