How to use rat tail collagen
1. Surface coating of cell culture vessels
Recommended concentration: 1-5 ug/cm2, with a coating concentration of 2 ug/cm2 as an example:
The collagen was diluted to 0.012 mg/ml with sterile 0.006 mol/L (0.36 g/L) acetic acid. Make sure the collagen solution is spread over the surface of the vessel and open on a clean bench overnight. It can also be used for 1 hour at room temperature, washed 3-4 times with PBS and used directly.
The coated containers can be stored for at least 3 months at 4-25 °C.
2. Preparation of three-dimensional collagen
The rat tail collagen type I can form a three-dimensional adhesive with a certain strength at a concentration of 1 mg/ml or more, and a gel concentration of 1-2 mg/ml is recommended. Collagen was dissolved in 0.006 mol/L acetic acid, and 0.06X volume of 0.1 mol/L NaOH was added during the gelation process to neutralize.
Required solution (both sterile, pre-cooled): 10xPBS (containing 10 mg/L phenol red for pH indication) or 10x medium, 0.1 mol/L NaOH, 0.1 mol/L acetic acid (usually not used), Double distilled water
A. Preparation of three-dimensional collagen without cells (for the preparation of 1 ml, 1 mg/ml three-dimensional gel as an example): 200 ul of raw rat tail collagen type I (5 mg/ml) was added to a centrifuge tube placed in an ice bath. , add 690ul H2O. It is then added to 12 ul of 0.1 mol/L NaOH (if 12 ul of 0.1 mol/L NaOH is added to the collagen solution in turn, local collagen condensation will occur due to NaOH not mixing quickly) and mix immediately. Add 100ul of 10xPBS or 10x medium, mix well and add to the culture vessel immediately. (The pH is about 7 after mixing. If phenol red is not added to PBS or culture solution, it needs to be tested with pH test paper for the first time.) The culture vessel was allowed to stand at room temperature (about 25 degrees) for 20 minutes to be solidified, and then transferred to an incubator. If 10x PBS is used in the formulation, an appropriate volume of cell culture fluid is required to be pre-equilibrated before use.
B. Preparation of three-dimensional collagen containing cells (for example, 1 ml, 1 mg/ml three-dimensional gel): Prepare cells suspended in the culture solution and place in an ice bath. Add 200 ul of rat tail collagen type I (5 mg/ml) to 12 ul of 0.1 mol/L NaOH (if 12 ul of 0.1 mol/L NaOH is added to the collagen solution in turn, localized due to NaOH not mixing quickly) Collagen condensation), mix immediately. Add 23ul of 10xPBS or 10x medium and mix well. (The pH is about 7 after mixing. If phenol red is not added to PBS or culture medium, it needs to be tested with pH test paper for the first time.) Add 760 ul of cell suspension and mix immediately before adding to the culture vessel. After the culture vessel was allowed to stand at room temperature for 20 minutes to be coagulated, an appropriate volume of the cell culture solution was added and transferred to an incubator for cultivation. Shanghai Chuangsai provides rat tail collagen type I, C20-200110-2ml, price: 280 yuan
Note: The rat tail collagen type I can be quickly gelled when the pH is neutral at room temperature, and should be kept as low as possible during the operation.
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