The overall goal of this procedure is to quantify snare mediated cell fusion events by activated expression of beta galacto. This is accomplished by first expressing v and t snare proteins ectopically at the surface of cost seven cells. The next step of the procedure is to combine the cells expressing V snare proteins with those expressing T snare proteins.
This results in snare mediated cell fusion and the activated expression of beta galacto. Ultimately, the cell fusion induced beta galacto activity is measured using a spectrophotometer. The main advantage of this technique or the existing methods like the microscopic SAL region assay, is that it can analyze multiple snare combinations.
Quantitatively, Though this method may be applied to understand the mechanism and regulation of snare mediated membrane fusion. It may also be applied to have throughput studies. Demonstrating the procedure will be Doc Na Hassan, a postdoc and David Humphrey, a technician from my lab The day before the transfection load.
24 well plates with 12 millimeter glass cover slips prepare two wells for each combination of cells expressing unique plasmids in the experiment on those cover slips. Seed 30, 000 cost seven cells cultured in DMEM supplemented with 4.5 grams per liter glucose and 10%FBS to transfect. The V cells add to each, well a quarter microgram of the TTA plasmid and one set of plasmids expression.
Flipped proteins of interest in this case flipped vamps to transfect the T cells add a quarter microgram of the plasmid encoding TRE lacks Z, and the complementary set of plasmids encoding the flipped proteins of interest. In this case, snap 25 syntaxin one and syntaxin four. Now transfect the cost seven with lipectomy according to the manufacturer's instructions and culture, the cells for 24 hours, 24 hours after the transfection fix the cells in supplemented para formaldehyde for 10 minutes after the fixation, rinse the cells three times with PBS plus plus, then block the cells with supplemented FBS for 30 minutes.
After the block, incubate the cells with the anti mic monoclonal antibody. Nine E 10 for 30 minutes after four washes with PBS plus plus incubate the cells for an hour with fitzy conjugated secondary antibodies at a dilution of one to 500. After four more washes with PBS plus plus mount the labeled cells in commercially available anti fade reagent.
The cells are now ready for confocal microscopy the day before transfection seed 1.2 million costs seven cells in each 100 millimeter tissue culture dish and 200, 000 costs seven cells in each well of six well plates for each plasmid combination to be analyzed. Prepare one, well culture the cells for 24 hours. The next day add the plasmids for the transfection for V cells transfect a plasmid expressing the flipped protein of interest and five micrograms of p tet off to the cells in each 100 millimeter culture.
Dish cot transfect control cells in a 100 millimeter culture dish with an empty vector and P te off at the same relative concentrations as the V cells. After all the plasmids have been added to the V cells and control cells, add tcom mycin to those wells to prevent the N glycosylation of vamps. In each well of the six well plates cot transfect the T cells with one microgram of PBIG with the complementary plasmids expressing proteins of interest.
Now transfect the cost seven cells with lip according to the manufacturer's instructions. Then culture the cells for 24 hours, 24 hours after the transfection, detach the V cells from their culture dishes with enzyme free cell dissociation buffer. Start by washing the cells once with 20 milliliters of PBS.
Then add five milliliters of enzyme free dissociation buffer. After a 10 minute incubation at room temperature, the cells will dislodge by sharply tapping the plate check that they have detached under microscope. If less than half the cells have detached, wait another five minutes and tap the plate again.
Continue the incubation in detachment buffer until more than half of the cells have detached. Then count the V cells with a hemo cytometer. Then resus the V cells at a concentration of approximately 480, 000 cells per milliliter in supplemented heis buffer DMEM.
Now add one milliliter of resuspended V cells to each well containing the T cells and return the plates to the incubator. After six, 12, or 24 hours of co culturing the cells, wash them with PBS and add lysis buffer. Next, scrape the cells from the wells and transfer them to tubes for centrifuging.
After centrifuging, transfer the supernatant to a new tube and measure the expression of beta galacto using a commercially available assay according to the manufacturer's instructions during that protocol, be sure to stop the cholera metric reaction after 90 minutes by adding one molar sodium carbonate to score the results. Measure absorbance at 420 nanometers on a spectrophotometer ter. When CO seven cells were transfected with flipped snare plasmids flipped snare proteins were expressed at the cell surface.
Flow cytometry was used to measure the expression levels of snare proteins at the cell surface. In order to compare their membrane fusion capacities, vamps needed to be expressed at the same level. Therefore, the concentration of each flipped snare plasmid used in the transfection was titrated and optimized for transfection.
Under such conditions, vamps 1, 3, 4, 5, 7, and eight and syntaxin one and four were expressed at the same level at the cell surface respectively, the neuronal snares that mediate synaptic exocytosis were used to test the feasibility of the assay. Indeed, when the V cells expressing vamp two and T cells expressing SYNTAXIN one snap 25 were combined, robust beta galacto expression was detected. However, when either vamp two or SNAP 25 were not expressed, only baseline beta galacto sase activity was detected, indicating that cell fusion and the expression of beta galacto sase rely on interactions of v and t snares overall SYNTAXIN one, snap 25, vamp one, vamp three, and vamp eight had comparable and the highest fusion activities.
In contrast, baseline beta GAL activity suggests the protein combination does not drive membrane fusion such as the case between vamp five and SYNTAXIN one. Snap 25. While performing the enzymatic fusion assay, it is important to remember to add the unica mycin to prevent the an glycosylation of esna proteins and add DTT to prevent the disulfide bond formation among esna proteins.
After watching this video, you should have a good understanding of how to perform the cell fusion assay.
