Editing SARS2 Pseudovirus (section)

== Titering ==

To determine viral titers, we used either flow cytometry (for viruses packaging the ZsGreen backbone) or a&nbsp;luciferase assay (for viruses packaging the Luciferase-IRES-ZsGreen backbone). A detailed titering protocol is&nbsp;described below and differences between these two readouts are noted:<br/>1. Coat a 96-well cell-culture plate with 25 uL poly-L-lysine per well (Millipore Sigma, P4707) according&nbsp;to the manufacturer’s protocol. Poly-L-lysine improves cell adherence and prevents cell disruption&nbsp;during infection.<br/>2. Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells per well in D10 media.<br/>3. The next day (12-24 hours post-seeding), count at least 2 wells of cells to determine the number of cells&nbsp;present at infection.<br/>4. Prepare serial dilutions of the viruses to be titered in a final volume of 150 uL D10 growth media.&nbsp;a. For ZsGreen virus, we started with a 1:5 dilution and made three 1:5 serial dilutions.<br/>b. For Spike-pseudotyped Luciferase_IRES_ZsGreen virus, start&nbsp;with undiluted virus and made three 1:3 dilutions. For VSV G-pseudotyped Luciferase_IRES_ZsGreen virus, &nbsp;start with a 1:50 dilution.

5. Gently remove the media from the cells and slowly add the virus dilutions.<br/>6. Add polybrene (Sigma Aldrich, TR-1003-G) to a final concentration of 5 ug/mL. Add 3 uL of polybrene diluted to 250 ug/mL to&nbsp; final infection volume of 150 uL&nbsp; .<br/>Polybrene is a polycation that helps facilitate lentiviral infection through minimizing charge-repulsion between the&nbsp;virus and cells [56].<br/>7. At 48-60 hours post-infection, collect cells for analysis:

*a. For flow cytometry:
**&nbsp;Look at the cells under a fluorescent microscope and select wells that appear ~1-10%&nbsp;positive. Harvest cells from these wells using trypsin and transfer them to a Vbottom&nbsp;plate or microcentrifuge tubes. Pellet cells at 300xg for 4 min and wash twice&nbsp;with 3% BSA in PBS. After the final wash, resuspend in 1% BSA in PBS and analyze&nbsp;&nbsp;via flow cytometry. Use a&nbsp;Becton Dickinson Celesta cell analysis machine with&nbsp;a 530/30 filter to detect ZsGreen in the FITC channel. Resulting FCS files can be analyzed using FlowJo (v10).
**Calculate titers using the Poisson formula. If P is the percentage of cells that are&nbsp;ZsGreen positive, then the titer per mL is: -ln(1 – P / 100) x (number of cells / well) /(volume of virus per well in mL). Note that when the percentage of cells that are&nbsp;ZsGreen positive is low, this formula is approximately equal to: (% ZsGreen positive&nbsp;/ 100) x (number of cells / well) / volume of virus per well in mL). Furthermore, the titers&nbsp;using even the Poisson equation will only be accurate if the percentage of cells that&nbsp;are ZsGreen positive is relatively low (ideally 1-10%).
*&nbsp;For luciferase:
**Thaw luciferase reagent&nbsp;(Bright-Glo Luciferase&nbsp;Assay System ,&nbsp;Promega, E2610)&nbsp;at room temperature.&nbsp;&nbsp;
**Prepare cells by removing 100 uL media from each well. Accounting for evaporation,&nbsp;this leaves ~30 uL of media in each well.
**Add 30 uL of luciferase reagent, mix well, and transfer all 60 uL to a black-bottom&nbsp;plate (Costar 96-well solid black, Fisher, 07-200-590).
**&nbsp;Incubate plate for 2 min at room temperature in the dark then measure luminescence&nbsp;using a plate reader. We used a Tecan Infinite M1000 Pro plate reader with no&nbsp;attenuation and a luminescence integration time of 1 second.
**Plot RLUs vs. virus dilution. Select an amount of virus for further assays where there&nbsp;is sufficient (>1000-fold) signal above virus-only background and a linear&nbsp;relationship between virus added and RLU.

[[File:Lentivirus Titering.JPG]]

The following protocol was developed to streamline neutralization assays with Spike-pseudotyped lentiviruses.<br/>This protocol can be performed with either human sera or plasma, or monoclonal antibodies. Note that for safety,&nbsp;sera or plasma should be heat-inactivated in a biosafety cabinet prior to use as described in subsection 4.6.&nbsp;

1. Seed a poly-L-lysine-coated 96-well plate with 1.25x104 293T-ACE2 cells (BEI NR-52511) per well in 50&nbsp;uL DMEM (2.5x105 cells per mL). Plan to infect this plate 8–12 hours post-seeding.

2. About 1.5 hours prior to infecting cells, begin preparing serum and/or ACE2 dilutions in D10:

<br/>a. In a separate 96-well “setup” plate, serially dilute serum samples leaving 60 uL diluted serum&nbsp;in each well. For the data in Figures 4A & 4C, we started at an initial serum dilution of 1:80 and did serial 2.5-fold dilutions, meaning each replicate of the assay requires 5 uL of sera. For&nbsp;ACE2 (Fig. 4) we started with a concentration of 200 ug/mL and did serial 3-fold dilutions.<br/>b. Add 60 uL of D10 to wells corresponding to virus only and virus plus cells control wells. Add&nbsp;120 uL of D10 to media only and cells only control wells. See Figure 5 for an example plate&nbsp;layout.

<br/>3. Dilute virus to ~2-4x106 RLU per mL. Add 60 uL of diluted virus to all wells containing serum dilutions&nbsp;and the virus only and virus plus cells control wells.<br/>4. Incubate virus and serum at 37 C for 1 hr.<br/>5. Carefully add 100 uL from each well of the setup plate containing the sera and virus dilutions to the&nbsp;corresponding wells of the plate of 293T-ACE2 cells.<br/>6. Add polybrene (Sigma Aldrich, TR-1003-G) as described in subsection 4.4 for a final concentration of 5&nbsp;ug/mL in each well.<br/>7. Incubate at 37 C for 48-60 hours before reading out luminescence or fluorescence as described in&nbsp;subsection 4.4.<br/>8. Plot the data. For the curves shown in Figure 4, we fit and plotted the data using the neutcurve Python&nbsp;package (https://jbloomlab.github.io/neutcurve/).

[https://www.protocols.io/view/pseudotyping-lentiviral-particles-with-sars-cov-2-br44m8yw https://www.protocols.io/view/pseudotyping-lentiviral-particles-with-sars-cov-2-br44m8yw]



HEK293T cells (2 × 106) were plated in a 100-mm tissue culture dish and transfected the next day when they were about 75% confluent with a combination of the following plasmids:

*9 µg of pLV-eGFP (a gift from Pantelis Tsoulfas (Addgene plasmid # 36083; [https://n2t.net/addgene:36083 https://n2t.net/addgene:36083]; RRID:Addgene_36083),
*9 µg of psPAX2 (a gift from Didier Trono (Addgene plasmid # 12260; [https://n2t.net/addgene:12260 https://n2t.net/addgene:12260]; RRID:Addgene_12260), and
*3 µg of pCAGGS-S (SARS-CoV-2)(Catalog No. NR-52310: BEI Resources)
**or VSV-G (a gift from Tannishtha Reya (Addgene plasmid # 14888; [https://n2t.net/addgene:14888 https://n2t.net/addgene:14888]; RRID:Addgene_14888) as control.



Polyethylenimine (PEI) reagent (Millipore Sigma, #408727) was used for transfection following manufacturer’s protocols. Next day, the cells were checked for transfection efficiency under a fluorescent microscope, indicated by GFP fluorescence. The supernatants from cell culture at 24 h were harvested and stored at 4 °C and more (10 ml) complete media (DMEM + 10% FBS) was added to the plates. The supernatant from cell culture at 48 h was harvested and combined with the 24 h supernatant for each sample. The combined supernatants were spun in a tabletop centrifuge for 5 min at 2000×g to pellet the residual cells and then passed through a 0.45 micron syringe filter. Aliquots were frozen at − 80 °C. New 293T cells plated in 12 well tissue culture dish were infected with the harvested virus (supernatant) with a dilution range of 102 to 107. Virus titers were calculated by counting the GFP positive cells in the dilution with 20–100 GFP positive cells.

MMLV pseudovirions were made in a similar way. HEK293T cells were transfected with the following plasmids:

*10 µg pLNCX-GFP,
*1.2 µg pCMV-tat-HIV,
*6 µg pJK3, and
*3 µg of pL-VSV-G (control) or
**pCAGGS-S (SARS-CoV-2).

Supernatants were recovered in a fashion similar to above.
[[Category:Virus]][[Category:Protocols]]