The aim of this procedure is to expose cells cultured in 3D to interstitial fluid flow, and to quantify the flow mediated effects on cell invasion. This is accomplished by first preparing a gel solution consisting of type one collagen and matrigel. The second step is to add a specified concentration of cells to the gel solution.
Next, the cell suspension is added to a 12 millimeter diameter, eight micron pore size transwell, and incubated at 37 degrees Celsius until the matrix gels. The final step in the assay setup is to add specific amounts of media to the transwells to create static and flow conditions. 24 hours later, the transwell membranes are fixed, stained, and visualized for the quantification of the number of invaded cells.
The main advantage of this technique over existing methods, such as microfluidic, interstitial flow chambers, is that it does not require pumps or specialized equipment, and it allows researchers to test multiple conditions or treatments easily and quickly. Though this method can provide insight into cellular migration and invasion, it can also be used or applied to study the effect of interstitial fluid flow on other relevant cellular behavior, such as protein expression, cell proliferation, and alterations in cell signaling. Demonstrating this procedure will be a LIMA two chaa.
A graduate student in my laboratory Begin this procedure by throwing a small aliquot of matri gel on ice at four degrees Celsius. Next, prepare the gel recipe with PBS sterile water, sodium hydroxide, matrigel, and rat tail type one collagen. Then mix the components of the gel on ice in the same sequence and incubate the final solution for one hour at four degrees Celsius.
Now place a 12 millimeter diameter eight micro po cell culture inserts into a 12 well plate using a pair of sterilized forceps. Then count the cells and resuspend them in serum free media at five times 10 to the six cells per milliliter, add 100 microliters of cell suspension to 900 microliters of gel solution. After that, mix them thoroughly by pipetting gently up and down.
Subsequently, add 150 microliters of the final mixture to each insert. Then transfer the inserts to a 37 degrees Celsius 5%carbon dioxide incubator for 30 minutes until the gel polymerizes. For the static condition, add 100 microliters of the serum free media on top of the gel and 1, 200 microliters microliters under the insert.
The fluid levels inside and outside the insert in the well should be approximately equal, resulting in a minimal pressure difference across the gel and no interstitial flow. For the flow condition, add 100 microliters of the serum free media under the insert and 650 microliters above the gel. At the same time, try to avoid creating any air bubbles beneath the insert as they will prevent cells from migrating through the membrane at these locations.
Then place the plate in a 37 degree Celsius 5%carbon dioxide incubator for 24 hours. In this step, add 500 microliters of one times PBS per well into a new 24 well plate for washing the inserts. Then remove the medium remaining in the upper portion of the flow trends wells.
Next, use cotton swabs to remove the gel from the inserts. Wipe the top of the surface of the membrane in order to remove non-VA cells. Then wash the inserts by placing them in the 24 well plate containing one times PBS for 15 seconds.
After that, remove the PBS and add 500 microliters of 4%para formaldehyde underneath each insert. Incubate them for 30 minutes at room temperature for fixing the trans migrated cells. Then remove the PFA and rinse them once with 500 microliters one times PBS to remove the residual fixative.
Next, add 500 microliters of 0.5%Triton X 100 solution beneath the inserts and incubate for 10 minutes at room temperature. To perme the cells, cut the membranes outta the insert using a razor blade. Then place them into 100 microliters of two microgram per milliliter DPI in one times PBS solution.
Being careful to place the underside of the membrane facing down. The DPI solution must be prepared a few minutes prior to use and kept in the dark. Now wrap the plate in aluminum foil.
Place it on a shaker at 150 RPM for 30 minutes of room temperature. Then wash the membranes in 500 microliters one times PBS and place them back on the shaker for 10 minutes. To remove free jy, repeat the wash another two times.
The next step is to place the membranes on glass slides with trans migrated cells facing up. Add mounting solution to the membranes. Then cover them with cover slips.
Afterwards, count the dappy stain to nuclei and calculate the number of invaded cells according to the accompanying manuscript. This figure shows the trans migrated MDA MB 4 35 s cells on the membrane. The invaded cells were fixed after the interstitial fluid flow invasion assay and stained with DPI and Alexa Fluor 4 88 conjugated foid in to facilitate counting of invaded cells.
This is the membrane under brightfield and here are the dappy stain nuclei. The Alexa Fluor 4 88 foid in stained with F actin are shown here. This graph shows that the invasion of MDA MB 4 35 s metastatic melanoma cells was significantly enhanced by interstitial flow.
After 24 hours, the results are normalized to the average of the static invasion condition and the mean from six cell culture inserts is presented. The difference between conditions is statistically significant with a P value of 0.003. Once mastered, the interstitial fluid flow assay can be set up in two hours, followed by a 24 hour incubation, and then two hours for fixing and staining the transform membranes.
Following this procedure, cells, proteins, or nucleic acids can be easily extracted in order to answer additional questions such as how gene and protein expression change in response to interstitial fluid flow. Since its development, this has become an essential assay for researchers studying the effects of interstitial flow on cancer cells.
