The overall goal of this procedure is to determine the ability of ovarian cancer multicellular steroids to invade a mesothelial monolayer. This is accomplished by first incubating red fluorescent protein expressing ovarian cancer cells in low adhesion culture plates to form multicellular steroids. The next step is to plate green fluorescent protein expressing mesothelial cells on fibronectin coated glass bottom culture dishes to form monolayers.
The final step is to transfer the ovarian cancer phs from the low adhesion plates to the plates containing the mesothelial monolayers. Ultimately fluorescence time lapse video microscopy is used to quantitate the relative ability of control and experimental ovarian cancer. SP to form a whole in the mesothelial monolayer.
The main advantage of this technique over existing endpoint assays is that a combination of fluorescently labeled cells and time-lapse video microscopy is used to monitor the dynamics of mesial clearance over time Prior to the formation of ovarian cancer phe, it is necessary to prepare low adhesion. 96 well round bottom culture dishes. To produce these culture dishes, add 30 microliters of poly hema solution to each well of a 96 well cell culture dish.
Incubate the 96 well plates in a 37 degree Celsius non humidified incubator for 24 to 48 hours to evaporate the ethanol, leaving a film of poly hema on each. Well this poly Hema film prevents cells from attaching to the bottom of the well, forcing the cells to grow in suspension. The red fluorescent protein expressing ovarian cancer cells in this experiment are cultured in 10%base medium, a custom cell culture medium when the low adhesion culture plates are ready.
Trypsin is a plate of ovarian cancer cells by first washing the plate with PBS, then adding one milliliter of trypsin and incubating the plate at 37 degrees Celsius four five minutes. After adding 10%base medium to quench the trypsin, transfer the cell suspension to a 15 milliliter conical polypropylene tube. Pellet the cells in a tabletop centrifuge at 900 RCF for three minutes, aspirate the supernatant and resuspend cells in 10%base medium.
After counting the cells using a hemo cytometer, adjust the concentration of cells to 100 cells per 50 microliters of 10%Base medium add 50 microliters of the uniformly suspended diluted cell suspension to each well of the 96 well poly hema coated culture dish incubate the 96 well plate in a 37 degrees Celsius cell culture incubator for 16 hours to allow the ovarian cancer cells to cluster together, forming a single multicellular steroid in each well. To begin this procedure, preco the wells of a six well glass bottom mat tech dish with fibronectin. Add two milliliters of fibronectin PBS solution to each well of the dish and incubate at B room temperature for 30 minutes.
The green fluorescent protein expressing mesothelial cells for forming a mesothelial cell monolayer our cultured in 10%base medium trypsin eyes a plate of mesothelial cells and spin down in a tabletop centrifuge at 900 RPM for three minutes. Aspirate the supernatant and resuspend cells in 10%Base medium, adjust to the desired concentration with 10%base medium. When the 30 minute fibronectin incubation of the mat tech dish complete wash the wells with two milliliters of PBS aspirate the PBS and plate six times 10 to the fifth mesothelial cells per well in each well of the six well mat tech dish.
Incubate the mat tech dish in a 37 degree Celsius cell culture incubator overnight to allow the mesothelial cells to attach to the dish and form a monolayer. To begin this assay, use a pipette to collect the ovarian cancer PHE from the 96 Well poly hema coated plate aspirate the medium from one well of the six well mat tech dish containing a mesothelial cell monolayer wash once with two milliliters of PBS, add all of the PHE from the 96 whale plate to one well of the mattec dish. This is approximately three times the number of steroid that are going to be imaged to account for PHE landing on the part of the dish that cannot be imaged.
Since we wanna image only one steroid per field of view, aggregation of the steroid must be avoided. Rock the dish gently from side to side to distribute the steroids evenly across the monolayer before they attach. Place the mat tech dish on the stage of an inverted widefield fluorescence microscope.
Capable of performing time lapse imaging for the duration of at least eight hours. Use a motorized stage to image multiple positions in the dish with multiple OID inter collation events. In a single experiment, the ovarian cancer cell PHE will settle to the bottom of the dish and attach to the mesothelial cell.
Monolayer collects G-F-P-R-F-P and phase images of 20 plus sphe monolayer interactions every 10 minutes for eight hours. In the mesothelial cell clearance assay, the RFP expressing ovarian cancer cell PHE will invade the GFP expressing mesothelial cell monolayer creating holes in the monolayer. The sizes of the holes are measured by tracing the black holes in the GFP images using element software or another suitable software such as Image J.The size of the hole is then normalized to the initial spheroid size by dividing the size of the hole at eight hours by the size of the steroid in the corresponding RFP image at times zero, the mesothelial cell clearance assay can be used to compare the invasion ability of ovarian cancer cell sphe that have been genetically modified to elucidate the molecular mechanisms by which ovarian cancer cell steroid clear.
The mesothelial monolayer in this example, the MESOTHELIAL clearance ability of O VCA 4 33 ovarian cancer cells. Phs that have attenuated expression of Talon one was compared to that of control OVCA 4 33 OIDs. The holes produced in the monolayer by the spreading PHE was measured and six positions from each group were averaged.
As shown in this graph, the average clearance area created by Talon one knockdown steroids was significantly smaller than the average area created by control steroids suggesting that Talon is required for mesothelial clearance by OVCA 4 33 ovarian cancer sps. This time lapse movie shows a control OVCA 4 33 red steroid invading a green MESOTHELIAL monolayer. This next time-lapse movie is of an OVCA 4 33 red steroid with attenuated expression of Talon one invading into a green mesothelial monolayer.
When compared with the previous movie, it is obvious that attenuation of Talon one expression in the steroids decreases mesothelial clearance ability. After watching this video, you should have a good understanding of how to monitor the interaction of ovarian cancer, multicellular steroids, and mesothelial cell monolayers over time using time-lapse video microscopy.
