The overall goal of this procedure is to investigate the function of extracellular vesicles released by the Malaria parasite infected red blood cells. This method can help answer key questions in the Malaria fields, such as how the parasites regulate parasite-host communication. The main advantage of this technique is the visualization of vesicular arbitage by indoterot cells under study of the regulatory function of the vesicular RNA by indoterot cells.
Demonstrating the procedure will be Mya Alondez, Smartin Bagu, and Bayo Babatunde, all grad students from my laboratory. In a micro-centrifuge tube add 100 micrograms of extracellular vesicles and one milliliter of phosphate buffered saline. Then centrifuge the extracellular vesicles at a maximum speed of 20, 000 times g for seven minutes to form a pellet.
Once the centrifugation is over, discard the supernatant without disturbing the pellet. Then dissolve the extracellular vesicle pellet in 100 microliters of dilumate C.To ensure complete mixing pipette it several times. Next, in a new micro-centrifuge tube, add four microliters of the PKH67 ethanolic dye solution to 100 microliters of dilmant C.Then vortex the mixture well.
Prepare the 40 micromolar 2XPKH67 dye solution just before the staining process. Then quickly combine 100 microliters of two x extracellular vesicular suspension with equal volume of the PKH67 ethanolic dye solution. Then pipette the mixture 10 times to ensure proper mixing.
After complete mixing, incubate the extracellular vesicular and PKH67 ethanolic dye solution for five minutes away from the light. In the course of incubation, keep vortexing the mixture three to four times. To stop the staining reaction, add 200 microliters of serum to the mixture and incubate for a minute for the excess dye to bind.
Next, wash the extracellular vesicles with phosphate buffered saline thrice. After washing, centrifuge the sample at 20, 000 times g for five minutes. Then dissolve the extracellular vesicular pellet in 100 microliters of phosphate buffered saline to attain a final concentration of one microgram per microliter.
Transfer the endothelial cells in one milliliter of complete endothelial cell growth medium to a poly-elithene coated cover slip. Allow the cells to grow a monolayer on the cover slip. After the monolayer is formed, remove the medium carefully and add one milliliter of fresh medium to wash the cells twice.
Then add 50 micrograms of PKH67 stained extracellular vesicles to the cover slip and incubate for four hours. After incubation, aspirate the medium. To remove all the unbound extracellular vesicles wash the cells with phosphate buffered saline thrice.
Great care should be taken during staining manipulation of the cover slips by using fine tongs to avoid cell loss and cover slip breakage. After washing, use three percent of paraformaldehyde solution in phosphate buffered saline to fix the cells for 15 minutes. Again, wash the cells with phosphate buffered saline thrice and add 250 microliters of 0.1 percent tritan x 100 in phosphate buffered saline to permeablize the cells and incubate at room temperature for five minutes.
Next, wash the cells with phosphate buffered saline thrice and add 400 microliters of blocking buffer to the cells. Then incubate the cells at room temperature for thirty minutes to prevent non-specific binding. After blocking, wash the cells once with phosphate buffered saline.
Then dilute five microliters of phalloiden stock solution in 200 microliters of phosphate buffered saline with one percent bovine serum albumine to prepare the staining solution for each cover slip. Then add 200 microliters of the staining solution on the cover slip and incubate at room temperature for 20 minutes. Once the incubation is over, wash the cells with phosphate buffered saline thrice.
Then use the final concentration of two micrograms per milliliter of hooksed 3342 in 200 microliters of phosphate buffered saline to counter-stain the DNA for 30 seconds. After staining, add 400 microliters of phosphate buffered saline to wash the cover slip twice. Then carefully lift the cover slip with the help of forceps and invert it on the top of a drop of antifade mounting media on a glass slide.
Use a tissue to gently remove any excess media and then seal the surrounding with nail polish. Great care should also be taken not to expose the cells to too much of the dye during microscopy to avoid cell bleaching. In a 24 well tissue culture insert with 0.4 micromolar pour size, seed endothelial cells.
Then let the cells grow in the culture for five days without being disturbed to form a differentiated monolayer. Keep changing the medium every second day. Once the monolayer has formed, seed 50 micrograms in one milliliter of extracellular vesicles on the upper chamber.
Then add 20 milligrams per milliliter of rotomine dextran to the top well and incubate at 37 degree Celsius with five percent carbon dioxide. Monitor the migration of the fluorescent dextran to the bottom of the well by measuring the flourescency mission from 40 microliters of medium avoquate. Then program the excitation wavelength at 544 nanometers and emission wavelength at 590 nanometers for a multi-label micro-plate reader.
Use a hemocytometer to count the number of cells. Then re-suspend the endothelial cells in complete endothelial culture medium and seed the cells in a 96 well plate in 100 microliters of endothelial cell growth medium. To achieve a concentration of 0.1 to ten micrograms per milliliter add 100 microliters of antibiotic containing medium.
Monitor the viability of the cells every second day using a 20 times objective under light microscope. Continue culturing the cells for another 10 to 14 days and replace the antibiotic containing medium every two to three days, depending on cell growth. Next, add 10 microliters of MTS reagent in each well and mix the reagent.
Incubate the 96 well plate at 37 degrees Celsius for four hours. After four hours, briefly agitate the plate on a shaker. Then read the absorbents of the treated and untreated cells using a plate reader at 490 nanometers.
One day before the lentiviral transduction, seed the endothelial cells in one milliliter of complete medium. Wait until the cells reach 50 to 80 percent confluency for lentiviral transduction Before opening the tube, spin the lentiviral suspension at 200 times g for 30 seconds to avoid spill over. Then add hexidimethryn bromide to the cells to adjust to a final concentration of eight micrograms per milliliter and gently swirl the plate.
Add the lentivirus to the cells and again swirl the plate gently for 30 seconds to ensure proper mixing. To increase the lentiviral transduction efficiency, centrifuge the cell-viral mixture at 300 times g for 45 minutes at room temperature. Then incubate the cell-viral mixture at 37 degrees Celsius over night.
The following day, discard the viral particle containing medium and replace with pre-warmed, fresh, complete culture medium. On the second day, start puromycin selection. Replace the complete culture medium with puromycin containing medium.
After puromycin selection, harvest the cells. Following manufacturers instructions, isolate RNA from the cells using an RNA isolation kit. Use a reverse transcription kit to isolate the complimentary DNA with the selected micro-RNAs.
Then set up the quantitative PCR reaction. To monitor the internalization of the extracellular vesicles by endothelial cells, PKH67 labeled green vesicles were analyzed using confocal microscopy. In the assay, faloudine and hookst 33342, which stains act in red and nucleide blue, respectively are used to track the translocation of the vesicles inside the endothelial cells.
Confocal images obtained show ready uptake of the vesicles by the endothelial cells after four hours. Next, to study the permeability of the endothelial cell monolayer diffusion capability of rodamine b isothyosionadedextran was analyzed. This assay shows that incubating the endothelial cells with 50 and 100 micrograms per milliliter of extracellular vesicles increased the endothelial monolayer permeability after two hours.
In the plot, the x-axis represents the concentration of the extracellular vesicles and the y-axis represents the fold changes in the endothelial permeability read at 590 nanometers. Next, to determine the sensitivity of the endothelial cells with puromycin treatment, a kill curve was plotted. The kill curve shows that as less as 0.15 microgram per milliliter of puromycin is sufficient to kill all of the cells.
Further, to determine the expression level of micro-RNA451a isolated from the endothelial cells, real time quantitative PCR was done. The bar plots show significant overexpression of micro-RNA451a transcript against the housekeeper gene U6.After its development, this technique paved the way for researchers in the field of infectious disease to explore the role of Evs in host interaction and cellular communication via transfer of genetic material. After watching this video you should have a good understanding of how to visualize EV uptake by recipient cells and how to investigate small including RNA function in the regulation of endothelial cells.
Don't forget that working with the Malaria parasites and human blood can be extremely hazardous and precautions, such as wearing gloves, lab coat, and working under a sterile hood should always be taken while performing this procedure.
