The overall goal of this simple, cell-based bioassay is to detect, quantify, and monitor the activity of members of the vascular endothelial growth factor family of ligins. This method can answer key questions in vascular biology, virology, cancer, and opthamology, where VEG-FR family ligins radiate key processes in these diseases. The main advantage of this assay is it allows evaluation of VEG-FM family ligins in terms of their capacity to bind and cross link their different receptors without requiring specialized epithelial cells.
Culture the IL3 secreting cell line WEHI-3D by pipetting five times ten to the sixth cells in the log face of growth into four to ten T175 flasks containing 50 millimeters of fresh culture medium. Incubate for seven days or until the cells have passed their log phase of growth by about 24 to 48 hours. Decant the fluid and cells from the flasks.
And spin at 1, 000 Gs for 15 minutes to remove cells and cellular debris. Then remove the top 90%of supernaytent and filter it through a 0.22 micron filter unit. Aliquot the conditioned medium into one milliliter aliquots for assay preparation.
50 milliliter aliquots for making culture medium to passage a factor dependent cell lines and 200 milliliter volumes for long term storage at 20 degree Celsius. Store the smaller volumes at 20 Celsius. Culture control BAF3 cells in culture media containing 10%WEHI-3D conditioned medium.
and VegFR2-EPoR-BAF3 cells in the same media plus one milligram per milliliter G418. Passage cells at one to 15 dilutions from cells growing in log phase. Harvest control BAF3 or VegFR2VPoR-BAF3 cells for mid log phase cultures.
By gently pipetting to remove the cells from the bottom of the flask centrifuge at 750 Gs for five minutes to recover cell pellet. Re-suspend the pellet in 10 milliliters of mouse tonicity phosphate buffered saline and centrifuge again at 750 Gs for five minutes. Repeat the wash two further times to remove medium containing IL3.
Wash the cells once using media without DEHI-3D conditioned medium. It is important to wash away culture medium containing IL3 to ensure that no additional growth factors remain, which might generate a false positive result or high background activity. After centrifuging and discarding the supernayten re-suspend cells in the same IL3 free medium at a concentration of 7.4 times 10 to the fourth cells per milliliter.
Finally mix equal volumes of trypan blue in PBS with a cell population and load into a hemacytometer. Count at least 100 cells. Cells that take up the dye are considered dead or dying.
It is critical that cells used for the assay have high viability to ensure they are able to respond their assay conditions. Use a well calibrated P20 automated pipet and autoclaved tips to add 1, 000 cells in 13.5 microliters of IL deficient medium to the wells of a 72 well plate. Take care to mix the cell suspension during aliquoting to ensure cell settling by gravity does not bias cell concentration.
Use a well calibrated P2 pipet to add 1.5 microliters of IL3 free media in triplicate to the control wells. Intrictery to mix. In the same manner add the 10%WEHI-3D conditioned medium and the 100 nanograms per milliliter VegFA no IL3 control to the desired wells.
Finally add 1.5 microliters of the test samples in triplicate. Fill any unused wells of the microwell plate with sterile water, PBS or medium. Place water soaked tissue paper in a gas permeable container to create a humidified chamber that allows gas exchange.
Incubate the assay plates in the chambers in a humidified atmosphere of 10%carbon dioxide for 16 hours. After 16 hours of incubation analyze the plates using a standard inverted phase microscope at 40 to 100x magnification. At this time it is possible to discriminate between positive and negative samples.
Wells without support of growth factors or with medium alone will have reduced numbers of round cells as well as dead or dying cells and cellular debris. Whereas cells incubated with medium containing IL3 or ligin for VegFR2 are large and translucent and there is no or minimal evidence of granularity in the cell cytoplasm or cell debris in the culture. Add 10, 000 washed cells in 135 microliters of IL3 deficient medium to the wells of a standard 96 well plate.
Use a calibrated P20 pipet to add 15 microliters of the test samples and controls to the wells. Incubate the mixture of cells growth factors and/or inhibitor agents as before for 48 hours. At the completion of the incubation period evaluate the number of viable cells in the wells using the methods outlined in the written portion of the protocol.
Here are represented results of an assay in which the VEGF-R2 EPoR BAF3 bioassay is used to quantify the activity of the three recumbent human ligins with specificity for VEGF-R2. VEGF-A165, VEGF-C, and VEGF-D. Activity was quantified in the presence of bevacizumab, an inhibitory monoclonal antibody to VEGF-A165 or in the presence of trastuzumab, a non neutralizing antibody.
Data have been normalized versus the response to VEGF-A165 alone. As expected bevacizumab blocked the effect of VEGF-A165 in the assay but had no effect on the activity of VEGF-C and VEGF-D. Cells exposed to IL3 were highly viable.
Whereas those exposed to no additional growth factor show poor viability. When attempting this procedure it's important to bare in mind that they're a range of controlling agents in conditioned media that need to be generated prior to the assay. This technique paved the way for resurgence in vascular biology, virology, cancer and opthomology to explore the role of VEG-FM in ligins.
After watching this video you should have a good understanding how to prepare the bioassay cells, to run the bioassay and to conduct the quantitative or semiquantitative analyses of VEG-FM ligin activity.
