Vegf analysis by elisa using
#DVE00, Quantikine Human VEGF Immunoassay.
Materials and methods:
PC-3 parental prostate cancer epithelial
cells (CRL-1435) were obtained from American Type Culture (ATCC, Manassas, VA)
and cultured in RPMI medium (GIBCO-BRL) without phenol red, containing 10%
fetal calf serum (FBS) [complete medium] per ml in 5% CO2 at 37oC.
The cells were split after every 3 days. The stable transfectants as described
below, PC3-15LOS (A17), PC3-15LOAS (A17.1) and PC3-Zeo (mock-transfected), were
grown in medium containing 50mg/ml Zeocin
(Invitrogen).
Vascular
endothelial growth factor (VEGF) analysis by ELISA
PC-3 cells (1.5 X 106)
were grown in complete RPMI medium
(containing 10% FBS) in triplicate for 2 days (asw grow slowly) until they were
80-90 % confluent. The growth
medium was removed, cell briefly washed with RPMI medium without FBS and equal
volumes of fresh RPMI medium containing 2% FBS was added and cells allowed to
grow further for 24 hours. The
medium is harvested and tested for VEGF by Quantakine human VEGF ELISA
according to the manufacturers instructions (R & D Systems, MN). Results were expressed as pg/ml of
growth medium. This experiment is
performed in triplicate twice. The final values of VEGF concentration
shown are subtracted values from control (i.e., RPMI medium containing 2% FBS).
For HCT-116
cells, plate at 1.0 X 106 cells per well of 6-well dish in 2ml
overnight in McCoy’s 5A medium (Zeocin not needed here even if stable
transfection). Plate triplicate of blank also for plate reader blank.
Wash and then
treat in 1ml of 2% FBS! And without phenol red can’t hurt plus 10uM
CaCl2! (from 1000X 10mM sterile stock) required for 15-LO-1 expression.
Treat in 1ml (or
smallest volume possible) for 24-hours in triplicate for ELISA.
Isolate media
for ELISA and test for VEGF by Quantakine human VEGF ELISA according to the
manufacturers instructions (R & D Systems, MN).
Add 100X
protease inhibitors then freeze at –70’C until ready to use. Save protein to
adjust for concentration. You may have to concentrate 5X or try treating with
50uM if does not work. Isolate cells for western blot just in case may need.
Also tty to
treat with vehicle, linoleic acid, and 13-S-HODE each at 30 then try 50uM.
See Dick
D’Augustine group for questions.