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BACKGROUND
Cells should be attached on the surface of culture plate, coverslips, and slides for immunocytochemistry and other experiments otherwise, washing and adding other solutions will detach cells easily that will spoil the experiment.
Gelatin, a mixture of heterogeneous, water-soluble proteins present in collagen and isolated by boiling the tendons, ligaments, bones, and relevant skin with water.1 Fibronectin is adhesive glycoproteins obtain in both the cell surface and in plasma.2 Gelatin and fibronectin has many applications.
Aim of this experiment is to learn how to coat cell plates with gelatin and fibronectin to attach cells for the experiments.
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REQUIREMENTS
Sample: Cell suspension
Apparatus: Plate – 60 mm
Filter – 0.2 µM
Culture hood
Cell culture incubator
Chemicals: Gelatin and fibronectin solution
Nuclease free water (NFW)
1X HBSS.
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PROCEDURE
Keep gelatin solution in 37°C for 10-15 min to dissolve completely, add 2% gelatin in NFW and mix it properly for the final solution. Filter the solution with 0.2 µM filter, add 8-10 ml of the solution in 60 mm plate and keep in 37°C for 2 hours. Discard the solution and keep in incubator to dry for 5-10 min (optional wash with HBSS before drying). Harvest cells after counting in the plate and you can find the well-attached cells in the plate surface. You can coat the coverslips and slides for the immunohistochemistry and further examinations.3
Fibronectin solution in supplied in 0.5 mg/ml sterile solution of CAPS saline buffer. Method for coating is same as gelatin.
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CONCLUSION
This experiment explain how to coat culture plate, coverslip, and slides with gelatin and fibronectin for the cell attachment.
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REFERENCES
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Gelatin solution from Sigma-Aldrich Company (sigmaaldrich.com).
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Fibronectin solution from human fibroblast from Sigma-Aldrich Company (sigmaaldrich.com).
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Kumar, A., Mishra, H.K., Dwivedi, P., Subramaniam (2015). Secreted trophic factors of Human umbilical cord stromal cells induce differentiation and neurite extension through PI3K and independent of cAMP pathway. Annals of neuroscience2015 Apr;22(2):97-106. doi:10.5214/ans.0972.7531.220208.
