Luciferase assays in 293 cells

Reagents*

1) 2x HBS
Reagent Final [] grams dry
HEPES, pH 7.05 50 mM 5.0
KCl 10 mM 0.37
dextrose 12 mM 1.0
NaCl 280 mM 8.0
Na2HPO4 (FW 141.96) 1.5 mM 0.1065 g
500 ml total, pH to 7.05
2) CaCl2
CaCl2 2 M 29.4 g/ 100 ml
*stock solutions may be prepared and frozen at -20 C

Cell Transfection

• Take a 100 mm plate of conflent 293 Cells.
• Split the cells into 2 of 100 mm plates with 10 ml of complete DME.
  Incubate the cells at 37 C for about 24 hours.
• Suck off the medium and add 3 ml of trypsin/EDTA to each plate.
• Count the cells using hemacytometer after all the cells are well seprated.
• Seed 5 X 105 cells/well in the 6-well plate in a volume of 3 ml of the medium.
• Incubate the cells at 37 C for 18 to 24 hours before transfection.
• Warm up all the DNAs, 2 M CaCl2 and 2 X HBS to RT.
• For each transfection, add 219 ul of H2O (sterile), 31 ul of 2 M CaCl2, 20 ng of NF-kB/Luc reporter construct ( or other Luc reporter construct), 40 ng of b-Gal expression construct, and other approprate DNA constructs. Total amount of DNA can be adjusted to 3 to 5 ug. It is always beter to make stock solution as long as you could.
• Add 250 ul of 2x HEPES slowly with bubbling for 15 seconds using pastual pipets and
  automatic pipettor.
• Add the ppt thus formed (should be slightly cloudy) immediately to tissue culture cells; add the precipitate dropwise so you do not disrupt the cells. A mild-moderate drop in pH of the medium is normal. Place cells immediately into incubator and incubate overnight.
• Change the medium at about 12 after transfection.

Leuciferase Assay

• About 48 hours after transfection, suck off the medium.
• Add 3 ml PBS.
• Suck off PBS as clean as possible.
• Add 250 ul of 1 X LYSIS BUFFER.
• Use P1000 to pipet all cells in each well into a new 1.5 ml microcentrifuge tube.
• Votex the cells.
• Wait for 5 minutes at RT.
• Put cells in - 80 C freezer and wait till all the cell lysates are frozen.
• Thaw the cell lysates in 37 C waterbath till all ice are melted (DO NOT OVER THAW).
• Spin the cell lysates in the microcentrifuge for 2 minutes at 14K rpm.
• Take 20 ul for luciferase assay.

b-Gal Assay

Reagent

Z Buffer: Per liter
16.1 g Na2HPO4.7H2O
5.5 g NaH2PO4.H2O
0.75 g Kcl
0.246 g MgSO4.7H2O
Z buffer with b-2ME : mix 27 ul of b-2ME with 10 ml of Z buffer.
ONPG (O-Nitrophenyl-b-D-galactopyranoside): 4 mg/ml in H2O
1 M Na2CO3

Procedure

• Spin down the cell lysate.
• Put 50 ul of cell lysate supernatant into a microcentrifuge tube.
• Add 500 ul of Z buffer with b-2 ME.
• Add 100 ul of ONPG (4 mg/ml) and Vortex.
• Incubate the tubes at 30 C till you see yellow color.
• Stop the reaction by add 250 ul of 1 M Na2CO3
• Read at OD=420 (visible light).