Protocol for PAI-luciferase assay for TGF-b
Procedure taken from Dan Rifkins’ Lab, NYU.
Mink king epithelial cells (MLEC’s-clone 32) are stably transfected with human plasminogen activator inhibitor-1 (PAl-1) gene fused to firefly luciferase reporter gene. The cells should be passaged at a dilution of 1/5 to 1/20 in DMIEM (high glucose) supplemented with 1% pen/strep, 10% FBS and 200 mg/m1 G418 every two-three days. Note that cells should be kept sub-confluent and passaged only 25-30 times (i.e. thaw out new cells every 1-2 month).
Assay
1.Sub-confluent PAI-MLEC cells are trypsinised and resuspended in l0 ml of DMEM media containing 10%FBS, 1 % pen/strep and 200 mg/ml G418. Centrifuge cells at low speed (3.5 on clinical centrifuge) for 5 min, remove media and resuspend cells in fresh media. Count cells and make a suspension of 1.6x105 cells/ml and plate 100 ml/well using a 96 well tissue culture dish. Incubate plated cells at 370C, 5%C02 for at least 3 hrs for optimal attachment. Alternatively, cells can be resuspended into a solution of 8 x 104 cells/ml and into 96 well plates at 100 ml/well and incubated overnight.
2.After 3 hrs or overnight incubation of cells, the serum containing media is aspirated and washed twice in 100 ml of assay media (DMEM/0.1%BSA, 1% pen/strep). Test samples, antibodies etc., serially? diluted either 2 or 3 fold (100 ml) can then be added directly to attached cells. Similarly, TGF-b dilutions (500 pg/ml to 156pg/ml; 100 ml/well made in assay media can be added to the transfected cells. Incubate samples and TGF-b standard overnight (or 14 hrs) at 370C, 5% CC2. Low levels of fetal calf serum (2%). do not interfere with the assay, however, it is important that TGF-b standard curve samples are prepared in similar media. If culture media is heated (800C 5 min) or acid activated to assess total TGF-b, dilute samples to 25%, 10% and 2% before assaying for luciferase.
3.After incubation, aspirate culture media from the cells, gently wash with 100 ml/wel1 of cold PBS (3 x). Lyse cells in 500 ml/well of l x lysis buffer (Analytical Lununescence Lab., cat # 1820) and incubate at 40C for 20 min. If longer stability is needed lysed cells can be stored in -700C for up to one month. However, after lysis of the cell transfer 40 ml of lysate to an opague 96 well plate (Dynatech Microlite1, flat bottom) and assay at room temperature at 20 min.
Modification
Since VES reduced cell number it is better to use 12 well plate to do this assay. The cell number or protein level need to be determined in order to normalize the luciferase activity See transient luciferase assay).