We are looking for diverse approaches to disassociating fin tissues from a fish. We are currently using TrypLE but we've got a feeling that spiking it with additional proteases may get the job done better/faster. Any suggestions/recommendations greatly appreciated.
Did you at any point try just trypsin-EDTA? TrypLE seems to have a much weaker effect on cells in my experience. (approx 3 years)
Trypsin is tolerated by many cell types; however it is desirable to avoid trypsin in proteomic studies and serum-free cultures. Based on the application and cell types trypsin is employed with various constituents and concentration. Some of the attributes are discussed below.
EDTA (disodium ethylenediaminetetraacetic acid): Adhesion molecules in the presence of calcium determine cell-cell and cell-matrix interaction. To weaken the interactions, EDTA is frequently included with Trypsin which chelates the divalent cations (Ca2+, Mg 2+).
Phenol red: Optimum activity of trypsin is achieved at pH range from 7 to 9 (5466615). At this range inclusion of phenol red gives pink color. Due to environmental conditions the pH of trypsin may turn acidic, giving orange color and renders trypsin less effective. By adjusting the pH to 7.4 – 7.6 with NaOH trypsin activity could be optimized.
Diluent: Concentrated trypsin is being solubilized or diluted in a buffered salt solution that contains no Ca2+ or Mg2+, such as Hank's Balanced Salt Solution (Product No H9394), which maintains pH and osmotic balance. Besides, some trypsin products have 0.9% NaCL as diluent instead of hanks balanced salt solution.
Source and form: The major source of trypsin is porcine, which is available either in the form of lyophilized powder or as in solution. To avoid animal or microbial products, recombinant bovine trypsin is also expressed in corn, called Trypzean solution (T3449).
Concentration: Based on the cell type and application trypsin is used in various concentrations. For strongly adherent cell lines, trypsin of 2.5 % to 0.25% (10X to 1X power) is used. While the studies which require cell surface protein integrity, lower concentrated (0.05% trypsin) solutions are employed.
Hi Sebastian,
Tryple or trypsin are very week in dissociating cells from such tissues. I work on retina organoids and even for dissociating cells from retina in mice, triple fails. However, 1% papain in HBSS containing 5 U/mL DNase for 10–30 min at 37 °C, and then were trituration in Papain inhibitor ovomucoid with 5 U/mL DNase works fine to dissociate the cells. It's worth giving a try for your case. I am providing link to help you with reagents and detailed protocol in which authors used this approach.
https://www.nature.com/articles/s41419-018-1049-0
Hope it helps!
Ajay