Hello, I'm IL OV to study the coordination of different lipid synthesis pathways to remember and biogenesis. It's useful to have an experimental system where membrane biogenesis of occur rapidly and induc. We found that phagocytosis of latex beats is particularly useful for such studies because it cells very quickly synthesize new membranes to replenish those that were lost as wrapping material early during the engulfment process.
In this video, my colleague Ong Zang will demonstrate procedures were used to study phagocytosis induced changes in gene expression using I Luciferase based report assay approach where, Hi, I'm X from the leverage of exon North in the Department of Molecular and the cellular biology at Harvard University. Today I will show you how to use a report gene assay to analyze the regulation of genes involved in membrane biogenesis after phagocytosis. So let's get us started.
For this procedure, we use the cultures of human embryonic kidney 2 93 cells. These cells are grown in 10 centimeter culture dish in medium A, which consists of the bales modified eagles egos medium or DMM supplemented with 10%fetal B serum or FBS and a cocktail of antibiotics. Using a standardization technique, the cells are detached from the bottom of the dish, countered and then plated on polylysine coated the 96 well plates where they are grown for 24 hours until beginning transactions.
For our transfect, we routinely perform all conditions in triplicate and the transfect, a total of 50 to 100 and nanogram of plasmid DNA per well. Plaid mixes should include at least the reporter construct expressing firefly luciferase and the control plasmid expressing renal lucifers from a constitutive promoter. To begin, prepare a mixture of serum frame and antibiotic frame DM EM with trans two N three transfection reagent use 10 microliter of DMM for each well to be transfected and at three microliter of trans 2 9 3 reagent per microgram of plasmid DNA.
For example, if 20 wells of each to be transfected with 50 nanogram DNA at three microliter of trans 2 9 3 reagent to 200 microliter of DM EM.The solution is mixed by repeatedly pipetting up and down and then left undisturbed at room temperature for at least five minutes. Next, the DNA is pipee into separate micro centrifuge tubes. The concentrations of our plus meter stock solutions are usually between 10 and 100 nanogram per microliter.
Once the DM M transit mixture has incubated for five minutes, it is transferred to the DNA tube and the solution is again mixed by pipetting. Then lets the samples stand at room temperature for 20 minutes. During the 20 minute intubation, remove the media from the 96 web culture plate and replace it with a 19 microliter of fresh medium A once the incubation is complete at the 10 microliter of the D-M-M-D-N-A translate to three mixture to each well.
The mixture should be pipetted directly into the existing liquid. Don't let the pipette tip touch the wall of the tube. It is also important to include three wells of cells that will be left unts inspected.
The average luciferase activity in this wells will later be subtracted as the background place the 96 well plate into the 37 degrees C incubator and grow the cells for 24 hours. Once the 24 hour incubation is complete, we proceed with inducing membrane biosynthesis, biosis of lax beads. To begin, prepare a suspension containing cell culture medium and lax bead.
To do this, prepare medium B, which is a one to one mix mixture of DM EM and hemps, F term medium plus antibiotics and 10%FBS. Then at the 0.75 micron lax beads at the concentrations of up to one milligram per milliliter. Of course, every experiment should include controls where no beads are added.
Next, remove the media from the 96 well culture plate and replace each well with the 100 microliter of the bead suspension. Once the suspension is added, spin the plate for two minutes at 1000 G.This helps concentrate the bead at the bottom of the well and facilitates phagocytosis. Next, capture the cells at 37 degrees C for six to 16 hours.
Depending on the conditions and the promoter used to drive luciferase increase the promoter gene expression can be detected after one to three hours. For certain experiments, it might be necessary to uncouple fal from lipid synthesis, for example, to study the effects of certain drugs on lipid synthesis without affecting particle in government. In such cases, incubate the cells with the beat for 30 to 16 minutes to allow cells to cyto the beads.
After this incubation, the cells are washed two times with the PBS to remove unincorporated beads. Then fresh media is added and then cells are incubated for six to 16 hours for report gene expression. Now we are ready to proceed with the ferous assays.
To begin the Lucifer assays prepare a xis buffer containing 20 million molar trees, HCL pH H 7.8 10%volume to volume glycerol and 0.5%volume to volume triton X 100 prior to use adequate amount needed for the experiment. Then at one microliter per milliliter of one molar DTT. Do not reuse the leftover DT T solution.
Then at a protease inhibitor cocktail to a final concentration of 0.5 to one times when using the dual glow kit for the first time, prepare the firefly lucifers reagent by transferring the entire contents of one bottle of dual glow lucifers buffer to one bottle of dual glow luciferase substrate. Divide the unused luciferase reagent into 10 millimeter quas and store at minus 20 degrees C.Once the luciferase reagent is ready, remove the 96 well plate from the tissue culture incubator and place it on ice. Next, remove the medial by aspiration, then add the 40 microliter per well of lysis buffer and leave the plate on ice for 30 minutes.
Next, qua 15 microliter of Lucifer reagent per well. In a white opaque 96 well plate, we use Perkin S optive plate 96. Then at the 15 microliter of cell ate and incubate the plate at room temperature for 10 minutes.
After 10 minutes, read the luminescence in a microplate reader. Next, perform the vanilla lucifers asay in order toum data for the control plasmid. To do this, prepare immediately before, use appropriate amount of luciferase substrate solution by adding one part dual glow, stop and glow reagent to 100 parts.
Dual glow, stop and glow buffer. Next, add 15 microliter of substrate solution to each. Well incubate the plate at room temperature for 10 minutes and then measure the luminescence again.
Please note that we modified the manufacturer's recommended protocol for these dual glow ferous assays. In order to reduce the amount of reagents required the pro sample, now the plate can be analyzed again using a plate reader. Once the data have been collected with the micro plate reader, they can be transferred to a spreadsheet program such as the Microsoft Excel for further analysis.
First, calculate the average from the three unresected wells and subtract that value from each of the data points. Next divide for each transfected well the value for firefly luciferase. Buy that for luciferase and then calculate then for each set of triplicate samples, calculate average, then the deviation and coefficient of variation.
In our example, we use the different concentrations of beads which have been plotted on the x axis against the ratio of firefly over vanilla lucres activity. As you can see, there is a dose dependent increase in this ratio. We have just shown you how to use a lucifers based report, gene assay to measure expression from the LDL receptor promoter in response to phagocytosis of latex speeds.
When doing this procedure, it's important to remember to be sure that the cells are only 80 to 90%the confluent as a time of transfection. So that's it. Thanks for watching and good luck with your experiments.
