Currently, I am crosslinking biglycan onto my scaffold and need to evaluate its bioactivity post-crosslinking. I noticed that MCE has previously tested biglycan’s activity by examining its inhibitory effects on the growth of NIH-3T3 and 3T3-L1 cells. If possible, could you kindly provide any recommendations or protocols for assessing its bioactivity in this context?Thanks!
Lucian Miles
Measured by its ability to inhibit the growth of NIH-3T3 cells. The ED₅₀ for this effect is 1.427 μg/mL, corresponding to a specific activity of 700.7708 units/mg. Measured by its ability to inhibit the growth of 3T3-L1 cells. The ED₅₀ for this effect is 5.9 μg/mL, corresponding to a specific activity of 169 units/mg. Reconstitute the protein with sterile water to 200 μg/mL. Then dilute with PBS to prepare different concentrations. Add 100 μL Biglycan protein to a 96-well plate so that the final concentrations in each well are: 0, 0.000041, 0.00041, 0.0041, 0.0082, 0.041, 0.082, 0.41, 0.82, 4.1, 8.2, 20.5, 41, and 820 μg/mL. Incubate the protein-coated 96-well plate at 37 °C, 5% CO₂ for 8 hours, then block with 0.1% BSA in PBS for 30 minutes. Remove the solution from the wells and wash twice with serum-free medium. Seed the two cell types (NIH-3T3 and 3T3-L1) at a density of 5,000 cells per well, in a volume of 100 μL per well. (For the outer wells of the plate, do not add cells—fill with an equal volume of PBS instead.) Incubate the cells at 37 °C, 5% CO₂ for 48 hours. After incubation, add 10 μL CCK-8 solution per well under light-protected conditions, and incubate at 37 °C, 5% CO₂ for 2 hours.
Hello Chloe,
HY-P7663 currently has two assay systems/methods as follows:
Experimental procedure:
The answer to this question is provided by MedChemExpress Technical Support.
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