The overall goal of this procedure is to identify signaling events induced and viable responder cells following their physical interaction with nearby dead cells. This method can help answer key questions in the fields of cell biology, pathology and physiology such as the influence that dead or dying cells exert on their live neighbors. The main advantages of this technique are that it is straightforward and inexpensive, and that it can be applied to study biologic effects induced in live cells following their interaction with neighboring cells undergoing apoptosis.
Demonstrating the technique will be Lanfei Feng, a technician in my laboratory. To begin this procedure after passing the BUNPT cells on to 100 millimeter vacuum gas plasma treated tissue culture dishes, grow them in culture medium A to confluence and humidified 5%carbon-dioxide atmosphere at 37 degrees Celsius. After that, rinse the adherent cell monolayer three times with culture medium B.Then, induce apoptosis by incubating the cells in culture medium B, containing staurosporine, a nonselective protein kinase inhibitor, for 3 hours at 37 degrees Celsius.
After 3 hours, collect the medium containing floating apoptotic cells that have detached from the monolayer. Subsequently, centrifuge this medium for ten minutes at 500 times G.Afterward, discard the supernatant, wash the pellet three times with culture medium B, before adding it back to the adherent cells. Following this washing step, detach the remaining adherent cells by adding 5 millimolar EDTA in 1X calcium and magnesium free DPBS.
Collect the medium containing detached apoptotic cells and put the detached cells with the floating apoptotic cells in a sterile 15 milliliter polystyrene centrifuge tube. Then wash the apoptotic cells three times by centrifugation for ten minutes at 500 x G.Followed by resuspension in ten milliliters of 1X calcium and magnesium-free DPBS per wash. After the last wash, suspend the apoptotic cells in fresh culture medium B at 5x10^6 cells per milliliter, before using them to stimulate the responder cells.
Alternatively, fix the washed apoptotic cells in 0.4 paraformaldehyde in 1X DPBS for 30 minutes, then wash them three times with culture medium B, and suspend them in it at 5x10^6 cells per milliliter before using them to stimulate the responder cells. After passaging the BUNPT cells on to 100 millimeter diameter sterile polystyrene vacuum gas plasma treated tissue culture dishes, grow them to confluence and humidified 5%carbon-dioxide atmosphere under permissive conditions. Then rinse the adherent cell monolayer three times with 10 mLs of culture medium B per rinse.
Subsequently, detach the cells by adding 5 mM EDTA in 1X calcium and magnesium-free DPBS. Collect the detached cells from the EDTA containing medium and add the cell suspension to a sterile 15 mL polystyrene centrifuge tube. Next, wash the cells three times by centrifugation for ten minutes at 500 times G and resuspension in 10 mLs of calcium and magnesium-free DPBS per wash.
After the last wash, suspend the cells in fresh culture medium B at 5x10^6 cells per milliliter. Following this, induce necrosis by heating the cells to 70 degrees Celsius for 45 minutes in a water bath, gently vortexing this cell suspension every ten minutes. After the induction of necrosis, incubate the cells for two hours in a humidified 5%carbon-dioxide atmosphere at 37 degrees Celsius, and gently vortex the cell suspension every 15 minutes.
In this procedure, grow the BUNPT cells in sterile 100 millimeter tissue culture dishes under permissive conditions, until they achieve about 85%confluence. Afterward, aspirate culture medium A and rinse the cells three times with 10 mL of culture medium B per rinse. Then, serum starve the cells in culture medium B for 18 to 24 hours in a humidified 5%carbon-dioxide atmosphere under non-permissive conditions, in order to induce quiescence.
Label a separate tissue culture dish of confluent BUNPT cells for the six experimental conditions for the next procedure. Now, aspirate culture medium B from the pre-labeled dishes of quiescent BUNPT responder cells. To prepare the 100 millimeter dishes in different conditions, add two mL of either culture medium B with no cells, the apoptotic cell suspension at 5X10^6 cells per milliliter in culture medium B, or the necrotic cell suspension at 5X10^6 cells per mL in culture medium B.Achieve a dead to responder cell ratio of one to one by adding to mL of dead cells at 5X10^6 cells per milliliter to a confluent 100 millimeter dish, which contains 10X10^6 cells.
Gently swirl the dishes. Then incubate them at 37 degrees Celsius in the humidified 5%carbon-dioxide atmosphere. After 30 minutes, stimulate the responder cells with either vehicle or 15 nMOL EGF, according to the pre-labeling of the culture dish.
Incubate the cells for 15 minutes at 37 degrees Celsius in a humidified 5%carbon-dioxide atmosphere. Afterward, wash away the dead cells by adding five millimeters of ice-cold DPBS. Swirl the dish and aspirate the wash fluid.
Repeat the wash three times. Then immediately place the responder cell dishes on ice for further processing. This figure shows that exposure of quiescent BUNPT responders to apoptotic cells for 30 minutes strongly inhibited basal phosphorylation of GSK3 alpha beta.
To prevent physical interaction between BUNPT responders and apoptotic cells, BUNPT responders were grown in a permeable support system, and separated from apoptotic targets by of 0.4 micron polycarbonate membrane. Prevention of physical interaction between BUNPT responders and apoptotic cells abolished the ability of apoptotic targets to inhibit phosphorylation of GSK3 alpha beta. To evaluate the role of phagocytosis, the cytoskeletal inhibitor Cytochalasin D was used to prevent phagocytic uptake of dead cells.
Inhibition of phosphorylation of GSK3 alpha beta in response to apoptotic cells occurred in the absence of phagocytosis. Once mastered, this technique can be done in 4-6 hours if it is performed properly, excluding the time required for gel electrophoresis and immunoblotting. While attempting this procedure, it's important to remember that using a cellular suspension as opposed to a soluble ligand as a stimulus poses several inherent and largely unavoidable experimental obstacles.
Using the same procedure, one can study other cell functions, such as survival or proliferation or cell growth in order to determine the functional consequences of the signaling events that are induced in live responding cells following their interaction with nearby dead cells. After watching this video, you should have a good understanding of how to identify specific signaling events that are induced in viable responder cells following their physical interaction with nearby dead cells.
