Luminescent β-galactosidase Detection Kit II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Pretreat cells with LPS. Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature	Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Upstream tips
Pretreat cells with LPS. Warm enough Reaction Buffer and Reaction Substrate for the entire experiment to room temperature
Protocol tips
Add 200 µl of the Reaction Buffer Mixture to each cell lysate and mix gently. Incubate at room temperature (20–25°C) for 60 min

Publication protocol

The mouse COX-2 promoter-luciferase construct pCOX-511, NF-κB promoter-luciferase construct pNFKB-Luc (Stratagene), and pSV40-β-galactosidase control vector (Promega) were generous gifts from Dr. Francesc Ventura (Universitat de Barcelona). Cells were transfected using Lipofectamine LTX (Invitrogen), and after 6 h the treated group was co-cultivated with MSCs. The next day, cells were serum starved before reaching confluence and treated with LPS (1 μg/mL; Sigma-Aldrich) for 24 and 48 h. Luciferase activities were quantified using the luciferase assay system (Promega, Madison, WI, USA). Luciferase values were normalized using β-galactosidase, and activity measured with the Luminescent β-Galactosidase Detection Kit II (CLONTECH, Palo Alto, CA, USA).

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