Biology Reference
In-Depth Information
Production of Adenovirus and Genetic Modification of
bMscs
AdenoVec-GFP-sFlt-1 and AdenoVec-GFP (InvivoGen, San Diego, CA) were
transfected into HEK-293 cells using jetPEI (Qbiogene, Nottingham, UK) ac-
cording to the manufacturer's protocol. Seven to 14 days later, the recombinant
adenoviruses were harvested from the cultures and the viruses were further ampli-
fied in 293 cells, and then tittered by plaque assay. BMSCs were infected with
adenoviruses at a multiplicity of infection (MOI) of 3000 for 2 hours followed
by replacing the infection medium. Twenty-four hours later, the virus-infected
BMSCs were harvested for subsequent experiments [11].
Western Blot Analysis
Western blot analysis was performed as described previously [13]. Briefly, the
secreted proteins in supernatants of the culture were precipitated by TCA-DOC/
acetone and the western blot assay was performed using standard method with
primary monoclonal antibody of anti-sFlt-1 (1:500 dilution, Santa Cruz Biotech-
nology, Santa Cruz, CA, USA) and a biotinylated secondary antibody, and the
proteins on the membrane were visualized by Vectastain ABC kit (Vector Labora-
tories, Burlingame, CA, USA).
Mouse endothelial cells Proliferation inhibition Assay
The mouse endothelial cells proliferation inhibition assay was performed as de-
scribed previously for examining suppression of sFlt-1 in the conditioned media
on VEGF-driven proliferation of endothelial cells [14]. The conditioned media
were obtained from BMSCs infected with Adv-GFP-sFlt-1, Adv-GFP, or PBS
(mock infected), respectively. Endothelial cells were grown in 24-well plates at
37°C in DMEM containing 10% FBS. At 50% confluence, the cells were washed
with PBS after removal of the DMEM media, and then 0.5 ml of conditioned
media was added. Fifteen minutes later, VEGF was added to each well with a final
concentration of 10 ng/ml and the cells were incubated at 37°C. After 72 h, the
cells were trypsinized, and the number of viable cells was counted using a trypan
blue assay.
Alginate encapsulation Assay
Alginate-encapsulated tumor cell assays were performed as described previously
[13]. Briefly, tumor cells, BMSCs expressing sFlt-1 or mixed cells were resuspended
Search WWH ::
Custom Search