Materials and Reagents

1. NIH3T3 cells or NIH3T3-Light2 cells (ATCC, catalog number: JHU-68 )
   Note: NIH3T3-Light2 cells were initially generated in Dr. Beachy’s Laboratory and were deposited in ATCC, the public available source of biomedical reagents including laboratory made cell lines. This cell line is available from Dr. Beachy’s lab per request.
   
2. Hh-conditioned medium
   
3. Fetal bovine serum (FBS)
   
4. Bovine calf serum (BCS)
   
5. DMEM (high glucose) (Life Technologies, Invitrogen^TM, catalog number: 11965 )
   
6. TransIT-2020 (Mirius, catalog number: MIR5404 )
   
7. 8XGli-Firefly luciferase expression construct
   Note: The 8XGli-Firefly luciferase expression construct is made at Dr. Beachy’s Laboratory. Eight consecutive repeats of Gli responding element (8XGli) are PCR amplified and cloned into the minimal enhancer region of pGL3-Luciferase [promega] vector. Map of pGL3 is available from promega.
   
8. pRL-SV40-Renilla luciferase expression construct
   Note: The pRL-SV40-Renilla luciferase expression construct is made at Dr. Beachy’s Laboratory.
   
9. Dual-Luciferase @ Reporter Assay System (Promega Corporation, catalog number: E1960 )
   
10. Growth media (see Recipes)
    
11. Shh-conditioned media (see Recipes)
    
12. Serum deprived media (see Recipes)
    

Equipment

1. 15 ml thermal scientific Nunc conical tubes
   
2. T75 culture flask (BD Biosciences, Falcon^®, catalog number: 353135 )
   
3. 24-well tissue culture plate (BD Biosciences, Falcon^®, catalog number: 353047 )
   
4. 96-well flat-bottom plate (Corning, Costar^® , catalog number: 3915 )
   
5. Centrifuge (Eppendorf, model: 8810R )
   
6. Water bath
   
7. CO₂ incubator
   
8. Berthold Luminometer (Berthold Technologies, model: Centro XS LB960 )
   

Procedure