Editing Spike Protein VLP (section)

=== Protocol ===
<div>
1. Cell Seeding for Pseudotyped Particle Production

'''NOTE:''' Perform this step in the biosafety cabinet.

#By standard cell culture techniques, obtain an 80–90% confluent 75 cm<sup>2</sup> flask of HEK-293T/17 cells passaged in complete Dulbecco's Modified Eagle's Medium (DMEM-C) containing 10% (vol/vol) fetal bovine serum (FBS), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 100 IU/mL penicillin, and 100 µg/mL streptomycin. Prepare DMEM-T medium for transfections (its composition is the same as DMEM-C but without antibiotics).
#Wash cells with 10 mL of pre-warmed (37 °C) Dulbecco's Phosphate Buffered Saline (DPBS) twice.<br/>'''NOTE:''' Handle HEK293T/17 cells with care as they easily detach.
#Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 °C. Place the flask of cells at 37 °C, 5% CO<sub>2</sub> incubator for 3−5 min or until cells start detaching.<br/>'''NOTE: '''Avoid incubating cells with trypsin for more than 5 min as this typically leads to cell clumping.
#Deactivate trypsin by adding 4 mL of DMEM-C medium and count cells using a cell counting slide and light microscope.<br/>'''NOTE:''' To avoid having to count too many cells, an additional dilution step may be required beforehand. Remember to factor in this dilution when calculating the actual cell density of trypsinized cells.
#Dilute cells to 5 x 10<sup>5</sup> cells/mL with DMEM-C.
#Seed 6-well tissue culture plate with 2 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion.<br/>'''NOTE:''' This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good transfection efficiencies and pseudotyped particle production.
#Incubate the plate overnight (16–18 h) in a 37 °C, 5% CO<sub>2</sub> cell culture incubator.

2. Three-plasmid Co-transfection

'''NOTE:''' Perform this step in the biosafety cabinet.

#Observe cells under an inverted light microscope to check for the cell morphology and density.<br/>'''NOTE: '''Ideally, cell density should be in the 40–60% confluency range. It is critical that cells are neither too confluent (80−90% confluent) nor too sparsely distributed (20−30% confluent) in each well. A cell density of 40−60% confluency will ensure good pseudotyped particles production.
#'''Plasmids mix'''
##Calculate the plasmid mix for each envelope glycoprotein following the quantities for one well of a 6-well plate shown in '''Table 1'''. Multiply quantities if transfecting several wells and include an extra well to avoid running out of mix.<br/>'''NOTE:''' Along with the SARS-CoV S and MERS-CoV S encoding plasmids, include empty vector control for the generation of negative control particles which lack viral envelope glycoproteins (Δenv particles) along with a positive control glycoprotein such as vesicular stomatitis virus (VSV) G glycoprotein that is known to robustly infect a very wide range of cells (VSV-Gpp). Plasmids are available upon request.
##Mix calculated volumes of plasmids and reduced serum cell culture medium (see '''Table of Materials''') in a microcentrifuge tube.
#'''Lipid-based transfection reagent mix''' (see '''Table of Materials''')
##Calculate the volumes for the transfection reagent mix from the quantities shown in '''Table 2''' for one well (1:3 transfection ratio, multiply quantities as needed). Include extra wells to avoid running out of transfection reagent mix.
##Mix calculated volumes of lipid-based transfection reagent (3 µL per well) and reduced serum cell culture medium (47 µL per well) in a microcentrifuge tube, making sure to add the transfection reagent into the reduced serum cell culture medium and not the other way around.
#Incubate both mixes (for one well: 50 µL of plasmids mix and 50 µL of lipid-based transfection reagent mix) separately for 5 min at room temperature.
#Add the contents of the transfection reagent mix to the plasmids mix at a 1:1 ratio (for 1 well: 50 µL of each mix)
#Perform thorough up-down pipetting of the resulting mix.
#Incubate the mix for at least 20 min at room temperature.
#Aspirate the spent medium of cells.
#Add gently 1 mL of pre-warmed (37 °C) reduced serum cell culture medium per well.
#Add dropwise 100 µL of transfection mix to each well.<br/>'''NOTE:''' Exercise care when adding the transfection mix to wells of HEK-293T as they detach easily.
#Incubate cells in a 37 °C, 5% CO<sub>2</sub> cell culture incubator for 4–6 h.
#Add 1 mL per well of pre-warmed (37 °C) DMEM-T medium, which does not contain antibiotics<br/>'''NOTE:''' This is a key step. Transfection reagents increase cell permeability and increase sensitivity to antibiotics. To ensure good transfection efficiency and pseudotyped particle production, it is important to avoid using cell culture medium containing antibiotics
#Incubate cells in a 37 °C, 5% CO<sub>2</sub> cell culture incubator for 48 h.

3. Pseudotyped Particles Collection

'''NOTE: '''Perform this step in the biosafety cabinet.

#Observe cells under inverted light microscope to check for cell morphology and general condition. Also check the color of the medium which should be light pink/slightly orange.<br/>'''NOTE: '''This is an important step. If there is too much cell death associated with the transfection or the medium color turned orange/yellow (acidic pH), this will typically be associated with lower yields in infectious pseudotyped particles
#Transfer supernatants of transfected cells to 50 mL conical centrifuge tubes.
#Centrifuge tubes at 290 x ''g'' for 7 min to remove cell debris.
#Filter clarified supernatants through a sterile 0.45 µm pore-sized filter.
#Make small volume aliquots (e.g''.,'' 500 µL or 1 mL) of pseudotyped virus solution in cryovials.
#Store at -80 °C.<br/>'''NOTE: '''The protocol can be paused here. Pseudotyped particles are stable at -80 °C for many months but once thawed, avoid re-freezing them as they will lose infectivity

4. Pseudotyped Particle Infection of Susceptible Cells

'''NOTE:''' Perform this step in the biosafety cabinet.

#'''Cell seeding of susceptible cells in 24-well plate'''
##Obtain by standard cell culture techniques 80−90% confluent 75 cm<sup>2</sup> flask of susceptible cells: Vero-E6 cells for SARS-CoV pseudotyped particles (SARS-Spp) and Huh-7 cells for MERS-CoV S pseudotyped particles (MERS-Spp).<br/>'''NOTE:''' To confirm whether the pseudotyped particles that have been produced are infectious, it is important to carefully choose an appropriate susceptible cell line for pseudovirion infectivity assays. Using poorly permissive cells will lead to low infectivity.
##Wash cells twice with 10 mL of pre-warmed (37 °C) DPBS.
##Aspirate the supernatant and detach cells with 1 mL of 0.25% trypsin solution pre-warmed at 37 °C. Place the flask of cells at 37 °C, 5% CO<sub>2</sub> incubator for 3−5 min or until cells start detaching.<br/>'''NOTE:''' Avoid incubating cells with trypsin for more than 5 minutes as this typically leads to cell clumping. Huh-7 cells are especially sensitive to this effect.
##Deactivate trypsin by adding DMEM-C medium and count cells using a cell counting slide and light microscope.
##Dilute cells to 5 x 10<sup>5</sup> cells/mL with DMEM-C.
##Seed wells of a 24-well plate with 0.5 mL of cell solution per well and gently move the plate back and forth and side to side to evenly distribute cells, avoiding circular motion<br/>'''NOTE:''' This is a key step. Evenly distributed cells will ensure that cells do not clump at the center of wells. In turn, this will ensure good infectivity assays. For each pseudotyped particle (SARS-Spp, MERS-Spp) and condition, prepare three wells for three experimental replicates. Include wells for the non-infected (N.I.), empty vector Δenv particles and positive control particles such as VSV-Gpp
##Incubate the plate overnight (16−18 h) in a 37 °C, 5% CO<sub>2</sub> cell culture incubator
#'''Pseudotyped particle infection'''
##Observe cells under light microscope and visually confirm that there is a confluent carpet of cells.
##Bring cryovials of pseudotyped virus to thaw on ice.
##Wash cells three times with 0.5 mL pre-warmed (37 °C) DPBS<br/>'''NOTE:''' This is a key step. Cells that are not properly rinsed prior to infection typically lead to poor infectivity readouts
##Aspirate the supernatants of cells.
##Inoculate cells with 200 µL of thawed pseudotyped particle solution.
##Incubate cells in a 37 °C, 5% CO<sub>2</sub> cell culture incubator for 1−2 h.
##Add 300 µL of pre-warmed (37 °C) DMEM-C medium.
##Incubate cells in a 37 °C, 5% CO<sub>2</sub> cell culture incubator for 72 h.

5. Infectivity Quantification by Luciferase Assay Readout

'''NOTE: '''Perform initial steps in the biosafety cabinet.

#Thaw luciferin substrate (stored at -80 °C) and 5x luciferase assay lysis buffer (stored at -20 °C) until they reach room temperature.
#Dilute luciferase assay lysis buffer to 1x with sterile water.
#Aspirate supernatants of cells infected with pseudotyped particles.
#Add 100 µL of 1x luciferase assay lysis buffer to each well.
#Place the plate on a rocker and incubate for 15 min with rocking at room temperature (from this point onwards the plate can be handled outside of a biosafety cabinet).
#Prepare microcentrifuge tubes for each well by adding 20 µL of luciferin substrate in each tube.
#Turn on the luminometer.
#Perform luciferase activity measurement one well at a time by transferring 10 µL of lysate to one tube containing 20 µL of luciferin substrate.
#Flick the tube gently to mix contents, but avoid displacing the liquid on walls of tube.
#Place the tube in device and close lid.
#Measure the luminescence value of the tube by using the luminometer.
#Record the relative light unit's measurement.
#Repeat steps 5.8–5.12 until all wells are analyzed.<br/>'''NOTE: '''With the appropriate equipment such as a plate reading luminometer, this process can be performed automatically. The assay will need to be scaled to the plate format (e.g.'','' 96-well plate format).

6. Data Analysis

#'''Calculation and plotting of relative luciferase units' averages and standard deviations'''
##Use a graph plotting software to calculate luciferase assay measurement averages and standard deviations of experimental and biological replicates.
##Plot data as bar chart with standard deviations.<br/>'''NOTE: '''When performing statistical analyses on data, make sure to include at least three biological replicates in data sets.
</div>
Subscription Required. Please recommend JoVE to your librarian.