Materials

• DMEM 10% fetal bovine serum
• L-ascorbic acid, add to medium at 50 ug/mL
• bovine chondrocytes
• XTT, 1 mg/mL in PBS (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide)

1. Thaw chondrocytes following recommended protocol (www.astartebio.com/thaw).
2. Adjust cell concentration to 1 million per mL.
3. Add 100 ul to each well of a 96 well plate.
4. Prepare mitogen, growth factor or test article in medium at twice the desired final concentration.
5. Add mitogen to wells at 100 ul per well. 
6. Incubate at 37oC for 1-3 days.
7. Remove 100 ul of medium from each well and add 50 ul of XTT per well
8. Incubate at 37oC for 2-4 hours.
9. Measure absorbance at 490 nm in a microplate reader.

We have observed peak proliferation at 2 days using FGF-18 as the stimulus. 

Culture supernatant collected in step 7 may be used to measure type II collagen production or proteoglycan synthesis. Baseline levels are very high under these culture conditions so increases may be harder to detect.