- Surface Coating :

Fibronectin : Calu-3 cells, like many other epithelial cells, often require a coated surface for optimal attachment. You might want to try coating the 6-well plate with fibronectin or collagen (e.g., collagen I or extracellular matrix proteins) to improve adhesion.

Poly-D-lysine : If fibronectin is not available, try coating the plate with poly-D-lysine, which can help enhance cell attachment.

- Cell Density and Plating Conditions :

Low Density : If you are plating too few cells, they may not adhere well or may undergo cell death. Try plating at a higher density (e.g., 50,000–100,000 cells per well) to ensure that they can adhere properly.

Cell Seeding : When seeding, ensure that the cells are well-dispersed across the surface. A uniform distribution can help with attachment and growth. Try gently pipetting up and down to break up cell clumps before plating.

-Trypsinization Protocol :

Trypsin Exposure : Prolonged exposure to trypsin (15 minutes) may damage the cells, making them more prone to detachment and death. Try reducing trypsinization time to 3–5 minutes or use a gentler dissociation method like Accutase or TrypLE, which are less harsh on cells.

Neutralizing Trypsin : After detachment, make sure to neutralize trypsin with medium containing serum as quickly as possible, to minimize any damage caused by the enzyme.

Hello,

Thank you for your reply. We will test the different methods you suggest. We have indeed considered an adhesion issue, since within the same flask, we can have cells on one side that are thriving while the other side is dead. My thesis supervisor ordered TrypLE for a gentler detachment. Are certain flask and plate references known to be more suitable for epithelial cells?

Tissue Culture Treated (TC-treated) Plates/Flasks.

Costar or Corning TC-Treated Plates / T-25 or T-75 flasks Brands like Costar (Corning) or Thermo Scientific Nunc offer standard TC-treated surfaces for multi-well plates, flasks, and petri dishes.

Use Low-Adsorption Plates for Long-Term Cultures : If your epithelial cells tend to produce excessive exosomes or extracellular vesicles, using low-adsorption plates (such as Greiner Bio-One's CELLSTAR® plates) might help reduce loss of proteins and other secreted molecules.

High Adhesion Plates for Tougher Cells : Some specialized plates, like Poly-D-lysine-coated plates, can help improve adhesion if cells remain challenging to attach even after using standard TC-treated vessels.

Check directly with your supplier.

Thank you for your advice, we are currently testing these methods. :)