I plan to conduct IF confocal microscopy of fixed and live cell experiments. I have used Poly -L- and -D- lysine (PDL). PDL lysine seems to work but I am still observing toxicity. Are there any other substitutions, such as collagen...?
You can overcoat the poly-lysine with fibronectin. some people dry and store the dishes;I do not dry them, but store for a few days with HEPES buffer In the refrigerator to use as needed. This protocol is from a non-fibroblast paper, but I use it in a cell bio class lab for fibroblasts. The poly-lysine allows more fibronectin binding.
”Culture Dish Preparation
Thirty-five millimeter polystyrene tissue culture dishes were coated with 100μg/ml poly- D -lysine in HEPES-buffered saline solution, pH 7.4. The dishes were incubated at 37◦C for at least 30 min, then rinsed twice with HEPES buffered saline pH 7.4.Fibronectin (75 μg/ml in HEPES buffered saline) was added to each dish and incubated at 37◦C for 1 h and rinsed with HEPES buffered saline. HEPES buffered saline contains 0.01 M HEPES, 0.01 M KCl, and 0.013 M NaCl in water, adjusted to pH 7.4. The dishes were rinsed with medium prior to addition of explants.”
Article Meningeal Foam Cells and Ependymal Cells in Axolotl Spinal C...
Hi Christopher Sarabia . Polylysines are unnatural adhesion factors that do not engage cellular integrins. Most adherent TC cells need to bind RGD containing cell adhesion proteins via integrins to survive.
Just coat the slides with 10% serum, which has vitronectin and fibronectin cell adhesion proteins that adhere to surfaces such as glass.
Hi Ellen A G Chernoff and Steingrimur Stefansson . Thank you both for the suggestion. I am studying a receptor at the plasma membrane, could Fibronectin interfere with receptor signaling at the PM or elicit other signaling pathways?
Hi Christopher Sarabia . Integrin signaling pathways have been well studied.
Article Integrin signalling at a glance
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