Editing MuLV Psueduvirus (section)

=== 4. Pseudotyped Particle Infection of Susceptible Cells ===

'''NOTE:'''&nbsp;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>&nbsp;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).

'''NOTE:'''&nbsp;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.
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<li>Wash cells twice with 10 mL of pre-warmed (37 °C) DPBS.</li>
<li>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>&nbsp;incubator for 3−5 min or until cells start detaching.</li>
</ol>

'''NOTE:'''&nbsp;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.
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<li>Deactivate trypsin by adding DMEM-C medium and count cells using a cell counting slide and light microscope.</li>
<li>Dilute cells to 5 x 10<sup>5</sup>&nbsp;cells/mL with DMEM-C.</li>
<li>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</li>
</ol>

'''NOTE:'''&nbsp;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
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<li>Incubate the plate overnight (16−18 h) in a 37 °C, 5% CO<sub>2</sub></li>
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