Evaluation of Extracellular Vesicle Function During Malaria Infection

Human RBCs were obtained from the blood of healthy donors, in accordance with the guidelines of Swissethics (swissethics.ch).

NOTE: P. falciparum parasite cultures (3D7) and EV production were previously described in Mbagwu, et al.¹¹ Because P. falciparum is a human pathogen, consult the local regulations for handling. The cultures should be kept sterile the entire time.

1. Fluorescence Labeling of EVs

NOTE: The following procedure takes advantage of the labeling technology to stably incorporate a green fluorescent dye (PKH67) with large aliphatic tails in the lipid region of the EV membrane. The reaction is performed in a 200 µL final staining volume containing a 20 µM final concentration of PKH67. Perform all steps at ambient temperature (20-25 °C)

1. Place 100 µg of EVs in 1 mL of PBS in a microcentrifuge tube.
2. Centrifuge the EVs in a microcentrifuge tube at maximum speed (20,000 x g) for 7 min to pellet.
   NOTE: Since hemozoin is present in EVs, a red-dark brownish pellet should be observed. If the supernatant is reddish, then the EVs have lysed.
3. After centrifugation, aspirate the supernatant carefully, in order to avoid removing any EVs.
   NOTE: Minimize the amount of remaining buffer present before resuspending EVs in diluent C in order to increase reproducibility and efficiency of the staining.
4. Resuspend the EV pellet in 100 µL of diluent C (2X EV Suspension) and gently pipet to insure complete dispersion.
5. Prepare a new microcentrifuge tube and add 4 µL of the PKH67 ethanolic dye solution to 100 µL of diluent C. Vortex well to mix. Prepare this 2x dye solution (40 µM) immediately prior to staining.
   NOTE: To avoid heterogeneous staining, do not add the PKH67 ethanolic dye solution directly to the EV pellet.
6. Rapidly, combine the 100 µL of 2X EV Suspension (step 1.4) with the 100 µL of 2X dye solution (step 1.5). Then, mix the sample by pipetting up and down 10 times.
7. Incubate the EV/Dye suspension from step 1.6 for 5 min protected from light and mix by vortexing 3-4 times during the 5 min incubation.
   NOTE: Incubating the EV/Dye suspension for longer times provides no advantage, because the staining is so rapid.
8. Stop the staining reaction by adding an equal volume (200 µL) of serum and incubate for 1 min to allow binding of excess dye.
   NOTE: Before stopping the staining, deplete the serum from EVs by centrifugation at 20,000 x g for 20 min.
9. Wash 3 times with PBS and spin down at 20,000 x g for 5 min. Resuspend the EVs in 100 µL of PBS to reach a final concentration of 1 µg/µL.

2. Visualize Uptake of EVs by Confocal Microscopy

NOTE: The following protocol describes the tracking of EV internalization by endothelial cells grown on glass coverslips. The endothelial cells are semi-immortalized human Bone Marrow Endothelial cells as described in¹².

1. Working in a sterile environment, coat coverslips with an excess of 0.01% poly-L-lysine for 10 min at room temperature (RT).
2. Aspirate the poly-L-lysine solution and allow the coverslips to dry completely.
3. Perform a viable endothelial cell count using Trypan blue exclusion and a hemocytometer.
4. Transfer 1 x 10⁵ endothelial cells in 1 mL of complete Endothelial Cell Growth Medium containing the Supplement Mix to the poly-L-lysine-coated coverslips and let the cells grow to a monolayer.
   NOTE: The medium contains growth factors that allow cells to form a monolayer and to form tight junctions.
5. Once the cells form a monolayer, carefully aspirate the medium and add 1 mL of fresh medium to wash the cells. Repeat the wash once more. Add 50 µg of PKH67-labeled EVs and incubate for 4 h.
   NOTE: To find the optimal time-point for uptake, a time-course can be performed.
6. After 4 h, aspirate the medium, and wash the cells 3 times with PBS to remove excess and unbound EVs. Fix the cells with 3% paraformaldehyde solution in PBS for 15 min.
   NOTE: Methanol can disrupt actin during the fixation process; therefore, it is best to avoid any methanol containing fixatives or other solvent-based fixatives. It is recommended to use methanol-free formaldehyde as a fixative.
7. Wash the cells 3 times with PBS. Add carefully 250 µL of 0.1% Triton X-100 in PBS to permeabilize the cells. Incubate at RT for 5 min.
8. Wash 3 times with PBS. Add 400 µL of blocking buffer (5% BSA in PBS) for 30 min at RT to block non-specific binding.
9. Wash the cells once with PBS. Prepare the staining solution by diluting 5 µL of the phalloidin stock solution in 200 µL PBS/1% BSA per each cover slip. Pipet 200 µL of the staining solution on the coverslip and incubate for 20 min at RT.
10. Wash 3 times with PBS. Counterstain the DNA for 30 s with Hoechst 33342 at a final concentration of 2 µg/mL in 200 µL of PBS.
11. Wash 2x the coverslip by adding 400 µL of PBS. Carefully grab the coverslip with forceps and invert it on the top of a drop of antifade mounting media on a glass slide. Remove gently the excess media with a tissue and seal each side with nail polish.
12. Store the slides protected from the light at 4 °C.
13. Visualize the cells using a confocal microscope at laser excitations 405, 488, and 543 nm and a 63X, 1.3 NA oil objective with fluorescent emissions at 450-470 nm (blue), 505-530 nm (green), and 620 nm.
    NOTE: Typical F-actin staining results are shown in Figure 1.

3. Endothelial Cell Permeability

NOTE: Endothelial cell permeability is assessed by measuring the transfer of rhodamine B isothiocyanate-dextran (average MW 70,000) across the endothelial cell monolayer. Dextran provides an excellent tool to study vascular permeability.

1. Count viable endothelial cells using 0.4 % Trypan blue exclusion and a hemocytometer.
2. Plate the endothelial cells at a density of 0.6 x 10⁵ cells to confluence in 24-well tissue culture inserts (0.4 µm pore size) and leave undisturbed for at least 5 days to form differentiated monolayers. Refresh the medium every second day.
3. Once the cells reach a monolayer, load the EVs (50 µg in 1 mL) on the upper chamber. Add the rhodamine-dextran to the top well at a final concentration of 20 mg/mL. Incubate the cells at 37 °C with 5% CO₂.
4. Monitor the transfer of the fluorescent dextran to the bottom well by measuring fluorescence emission from 40 µL medium aliquots. Set up the multi-label microplate reader at 544 nm excitation and 590 nm emission for measurements.
   NOTE: The amount of diffused dextran can be quantified using calibration curves established with the stock solution. In addition, kinetic experiments can be performed by removing small samples of the outer chamber medium during a time course (Figure 2). Even without any treatment, the dextran will diffuse through the cell monolayer.

4. Determine Puromycin Sensitivity

NOTE: Before transducing the cell lines with lentivirus, it is important to determine the kill curve of a selected drug for that particular cell line. To determine the minimum amount of drug concentration necessary to kill all of the cells, perform a dose response experiment using incremental doses of the selected drug. Because each mammalian cell line has a different sensitivity, before experimentation, the optimal concentration of the antibiotic should be determined by developing the kill curve titration as detailed below.

1. Count the cells using the hemocytometer. Resuspend the endothelial cells at a concentration of 1 x 10⁵ cells/mL in complete Endothelial Cell Growth Medium containing the Supplement Mix.
2. Plate 1 x 10⁴ cells into a 96-well plate in a final volume of 100 µL of the Endothelial Cell Growth Medium.
3. Add 100 µL of medium containing the antibiotics to obtain a concentration of 0.1-10 µg/mL (see Figure 3).
4. Examine the cell viability every 2 days under a light microscope using a 20X objective. Culture the cells for 10-14 days and replace the media containing puromycin every 2-3 days depending on cell growth.
5. Add 10 µL/well MTS Reagent into each well, mix, and incubate for 4 h at 37 °C in standard culture conditions.
6. Shake the plate briefly on a shaker and measure absorbance of treated and untreated cells using a plate reader at 490 nm (Figure 3).

5. Transduction of Endothelial Cells with Lentiviral Vector

1. Plate 1 x 10⁵ endothelial cells in 1 mL of complete medium the day before transduction. Perform transduction when the cells have reached 50-80% confluency.
2. Thaw the lentivirus on ice, and make aliquots of the viral stocks to avoid freeze-thaw cycles. Spin down the material at 200 x g for 30 s, in tubes before opening to avoid spill over.
3. Add hexadimethrine bromide to the cells at a final concentration of 8 µg/mL.
   NOTE: Hexadimethrine bromide helps the transduction efficiency, however in some cases it might be toxic for the cells. Therefore, the optimal concentration should be determined by performing a killing curve before the transduction.
4. Swirl the plate gently to mix. Add the appropriate quantity of viral particles (between 0.5-2 x 10⁶ particles) at a suitable multiplicity of infection (MOI) and swirl the plate gently for 30 s to mix.
5. Centrifuge the cell-viral particle mix in the centrifuge at RT for 45 min at 300 x g to increase the transduction efficiency. Incubate the cell-viral particle mixture at 37 °C overnight.
6. Remove carefully the viral particle-containing medium and discard appropriately. Replace it with fresh, pre-warmed complete culture medium.
7. Start the selection with puromycin on the second day: remove the medium and replace it with fresh medium containing the correct concentration of puromycin as determined previously in section 4 (Figure 3).
8. Harvest cells after puromycin selection and isolate RNA using an RNA isolating kit following the manufacturer's instructions.
9. Perform cDNA synthesis with the selected miRNAs using a reverse transcription kit (see the Table of Materials).
10. Set up the qPCR reaction according to the cited protocol¹³.