Growth, detection, quantification, and inactivation of SARS-CoV-2 (2024)

2.1. Materials needed

Chosen cell type (Vero-CCL81, Vero-furin, Vero E6, Vero-TMPRSS2, and MA104cells)

Standard media for chosen cell type (see Recipes)

Infection media (see Recipes)

SARS-CoV-2 seed stock

150cm² (T150) tissue culture flasks

15mL disposable polystyrene conical tubes with screw caps (e.g., Falcon)

50mL disposable polystyrene conical tubes with screw caps (e.g., Falcon)

1.5mL or 0.5-mL O-ring tubes

• 1.)

  In a standard BSL2 laboratory, plate cells for infection one day prior into two T150 flasks in standard media for the chosen cell type. One flask serves as a mock-infected control and the other for infection. Plate cells so they will be ~80–90% confluent the following day. *For instance, plate 1 x 10⁷Vero CCL81cells per T150 flask. Place flasks in a humidified 37°C incubator with 5% CO₂ overnight.

• 2.)

  Transfer flasks into BSL3 facility the following day. Rapidly thaw a SARS-CoV-2 stock at 37°C. Calculate the volume of virus needed to infect at the desired multiplicity of infection (MOI) using the following formula:

• 3.)

  Add the volume of virus calculated above to 20mL of infection medium.

• 4.)

  Remove medium from T150 flasks. Replenish with 20mL of fresh infection medium for mock-infected flask. Add 20mL of infection medium containing virus from step #3 to flask for infection.

• 5.)

  Incubate for 48–72hat 37°C monitoring daily for evidence of cytopathic effect (CPE) (Fig. 1). Use the mock-infected flask as a control for subtle CPE.

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  Fig. 1

  SARS-CoV-2 causes cytopathic effect on Vero E6 cell monolayers. Vero E6 cells were inoculated with SARS-CoV-2 at an multiplicity of infection (MOI) of 0.01 plaque forming unit (PFU)/cell and monitored for cytopathic effect at the indicated timepoints. Images were collected using an EVOS XL Core Imaging System. Magnification is 10X for all images.

*Harvesting at 48–72h post-inoculation has yielded the best titers in our hands; although, titers remain roughly the same when incubated for longer periods (4–5 days). CPE should be apparent by day 3 in Vero or MA104cells.

• 6.)

  To harvest virus, collect the cell culture supernatant by pipetting the media into two 15mL conical tubes. Centrifuge at 450×g for 5minat 4°C to clarify supernatants and pellet cell debris. Combine the supernatant from all tubes into a single vessel and gently mix using a serological pipette to ensure hom*ogeneity across aliquots of the stock. Pipette the supernatant into small aliquots (200–500μL) in O-ring tubes. Store at −80°C.

Real-time PCR assay for SARS-CoV-2 detection. Detection of viral RNA by reverse-transcription quantitative polymerase chain reaction (RT-qPCR) using a TaqMan probe is a highly-sensitive and specific method for measuring viral burden in a variety of specimens. Because CoVs generate subgenomic RNAs as a template for translation, the abundance of viral RNA varies for each gene and depends upon the gene position within the genome. Genes located closer to the 3′ end of the (+) sense genome will have a greater abundance of transcripts than those located at the 5′ end of the (+) sense genome. This should be considered when designing primer/probe combinations, as “N gene” transcripts will be more abundant than genomic RNA copies, which can be quantified by targeting sequences within the ORF1a gene. Many primer/probe combinations have been designed and validated, several of which are used in clinical diagnosis (CDC, 2020; Corman et al., 2020). In the clinical setting, precise copy-number quantitation of viral RNA is not necessary and instead sensitivity is paramount. However, quantitative assays are desirable for research applications, and may have utility in longitudinal studies of infected human subjects. RT-qPCR cycle threshold (Ct) values can be converted to transcript or genome copy number equivalents by generating an RNA standard curve, the design and production of which is described below.

2.2. Design of the primer/probe combination

The CoV replication strategy should be considered when designing a RT-qPCR assay. Primer/probe combinations targeting the N gene are most sensitive; those targeting the spike gene can also be used to titer spike-containing pseudoviruses; those targeting the ORF1a gene provide genome equivalents; and those targeting the leader sequence can give an estimation of the total number of viral transcripts (Table 1). For a given viral gene target, a template (~500–1000 bp) for in vitro transcription can be generated by RT-PCR using primers that flank the intended target, with the forward (F) primer also including a 5′ T7 promoter sequence (Vogels et al., 2020). If multiple targets are desired, a single dsDNA fragment can be synthesized to include concatenated gene fragments, each of which spans the entirety of the target amplicons. This strategy also can be used to quantify host genes of interest (e.g., ACE2).

2.3. Construction of the RNA standard

• 1.

  (Day 1) The DNA fragment/amplicon containing the primer/probe targets to be used in the RT-qPCR assay should be introduced into a vector containing a T7 (or other DNA-dependent RNA-polymerase) promoter sequence using Gibson Assembly, restriction enzyme cloning, blunt-end ligation, or gene synthesis. These vectors should be transformed into competent E. coli (e.g., DH5α) for antibiotic selection.

• 2.

  (Day 2) Pick clones and amplify to miniprep scale. We normally pick 6 to 12 clones to ensure proper cloning.

• 3.

  (Day 3) Purify plasmid from clones, and identify a clone with the proper insert using restriction enzyme digestion and/or Sanger sequencing.

• 4.

  (Day 4) Linearize ~2–4μg of the DNA in preparation for in vitro transcription by performing an overnight restriction digest using a high-fidelity restriction enzyme that cuts each plasmid only once in a position 3′ to the insert. The distance between the T7 transcriptional start-site and the 3′ end restriction site should be ~500–1500 nucleotides.

• 5.

  (Day 5) Run the linearized product on a 1% agarose gel. A shift in fragment size should be apparent relative to the non-linearized plasmid. Extract and cleanup the linearized product with a commercially-available gel-extraction (e.g., Qiagen) kit.

• 6.

  Perform in vitro transcription using a commercially available kit (e.g., MEGAscript T7). Note: to prevent contamination of PCR workstations with transcribed RNA, all steps hereafter should be performed in a contained hood/workspace that is separate from the area where PCR reaction setup is performed.

• 7.

  Digest DNA with DNase, then perform RNA cleanup using a commercially available kit (e.g., MEGApure).

• 8.

  Quantify the RNA using a spectrophotometer (e.g., Nanodrop or Qubit) by diluting the RNA with RNase-free water until the concentration is within the analytical measurement range of the spectrophotometer.

• 9.

  Calculate the copies of RNA transcript within each μL:

Note: the molecular weight can be calculated online (e.g., OligoCalc website).

• 10.

  Dilute the transcript with RNase-free water containing 1% of added ribonuclease inhibitor (e.g., RNaseOUT) to obtain 1–2mL of standard at a 1×10¹⁰ copies/μL. Mix by pipette.

• 11.

  Aliquot the diluted RNA transcript into PCR strip tubes (with individual caps) in aliquots of 6–12 μL/aliquot.

• 12.

  See Also

  Determining Viral Titer as Plaque Forming Units (PFU)Viral Concentration Determination Through Plaque Assays: Using Traditional and Novel Overlay Systems

  Freeze at −80°C. The remaining concentrated RNA can be frozen and re-quantified later as needed.

2.4. Validation and use of the RNA standard

The RNA standard is concentrated and poses a risk for contamination of reagents and specimens. Follow best-practices for PCR preparation (Standards Unit, 2010) and only handle RNA standards after all reagents and specimens have been stored. Appropriate no-template controls must be used to eliminate and track possible contamination. Wipe down work areas and pipettes with 10% bleach followed by 70% ethanol. Bleach pipette tips.

• 1.

  Create a 20x stock of primer/probe mix by diluting primers to a concentration of 10μM and probe to a concentration of 2μM.

• 2.

  For “n” number of reactions, create a master-mix for n+1 by combining one-step RT-qPCR reaction buffer, primer/probe mix, and reverse-transcriptase enzyme at the appropriate concentration/volumes. Aliquot master-mix into wells of a RT-qPCR-compatible plate.

• 3.

  Separate a single tube containing the RNA standard from the stock. Work quickly to avoid thawing other aliquots in the adjacent strip tubes.

• 4.

  Thaw the aliquot and briefly centrifuge to collect contents at the bottom of the tube.

• 5.

  Dilute the standard into a volume of RNase-free water to obtain 1.0×10⁹ RNA copies per reaction. Mix gently but thoroughly by pipette. Change gloves.

• 6.

  Make 10-fold serial dilutions in a PCR strip-tube by transferring 10μL into 90μL of RNase-free water. Mix each dilution thoroughly with a p100 pipette set to 70μL. Discard tips between each dilution.

Note: When testing a new RNA standard, perform serial dilutions several-fold below 1 copy per reaction. Reactions containing less that 1–10 copies/well should fail to amplify.

• 7.

  Transfer the appropriate volume of RNA standard from each dilution into the reaction plate using a multichannel pipette.

• 8.

  Perform real-time PCR using the following thermocycling parameters:

  • 1.

    48°C for 15min

  • 2.

    95°C for 10min

  • 3.

    95°C for 15s

  • 4.

    60°C for 1min – Acquire Signal

  • 5.

    Go to "step 3″ 49x (i.e., 50 cycles)

Note: these parameters may vary depending on the specific RT-qPCR kit used; our parameters have been tested using the TaqMan RNA-to-CT 1-step kit (Applied Biosystems) on the QuantStudio 6 flex Real-time PCR system (Applied Biosystems).

• 9.

  Upon completion of the run, examine your standard curve. Approximately 3.3 Ct should separate each dilution, which corresponds to a change of one log₁₀ copies for a reaction that is >90% efficient.

Quantification of SARS-CoV-2 by plaque assay. The plaque assay is the gold standard test for quantifying infectious virus in a sample. The plaque assay measures “plaques,” which describe the zone of cellular death that occurs after one infectious unit has entered a cell and spread to adjacent cells over the time period of incubation (Fig. 2). The assay does not rely on the use of any virus-specific reagents, which is beneficial when reagents are unavailable. As this cell-based assay typically is performed in 6-well plates, it is relatively low-throughput, labor-intensive, and may not be reliable when the samples themselves are cytotoxic (e.g., hom*ogenate from certain tissues) or when the virus is poorly cytopathic in a given cell type. Thus, it is important to choose a highly permissive cell type (e.g., Vero E6 cells) for which SARS-CoV-2 causes substantive cell death.

2.5. Materials needed

Vero E6 or Vero-furin cells

Vero cell culture medium (see Recipes)

Infection media (see Recipes)

Virus to be titered

96-well U-bottom plates

6-well or 12-well tissue culture plates

2X MEM+4% FBS (see Recipes)

2% methylcellulose (see Recipes)

4% paraformaldehyde solution (in PBS)

Crystal violet staining solution (see Recipes)

• 1.

  Plate approximately 7.5×10⁵ Vero E6 or Vero-furin cells/well into 6-well plates. Plate enough wells to test each dilution in duplicate (starting from 10⁻¹ to 10⁻⁶; 10-fold dilutions). Incubate cells overnight (12–18h) at 37°C.

*12-well tissue culture plates also will work. Plate approximately 2.5×10⁵cells/well.

• 2.

  Dilute samples to be titered in infection media in 96-well U-bottom plates. Make a 10-fold dilution series, providing enough volume to add 200μL per 6-well plate.

• 3.

  Remove existing cell culture media from 6-well plates. Add 200 μL of each dilution to one well of a 6-well plate (200uL to 12-well plate) starting with most diluted so the same pipette tip can be used up the dilution series.

• 4.

  Incubate 6-well plates at 37°C in 5% CO₂ for 1h, rocking plates every 15min to prevent cells from drying out.

• 5.

  Meanwhile, mix 2X MEM + 4% FBS with 2% methylcellulose in a 1:1 ratio. Place in 37°C incubator while plates are incubating to decrease viscosity of the solution.

• 6.

  After 1h incubation, add 2mL of MEM:methylcellulose mixture to each well of the 6-well plates (1mL–12-well plate).

• 7.

  Incubate plates at 37°C in 5% CO₂ for 3 days.

*After 3 days, plaques should be visible by eye when carefully held up to the light. If plaques are too small to discern, plates can be incubated for an additional day.

• 8.

  On day 3, gently remove methylcellulose overlays with a pipette and fix cells by adding 3mL of 4% paraformaldehyde (PFA) in PBS to each well. Incubate at room temperature for 20min.

• 9.

  Remove 4% PFA into an appropriate hazardous waste container. If this concentration of PFA has been approved as a method of SARS-CoV-2 inactivation by the Institutional Biosafety Committee, plates can be removed from the BSL3.

• 10.

  Add 1mL of 0.05% (w/v) crystal violet in 20% methanol to each well. Incubate for 20–30min. Remove crystal violet with a pipette and wash twice with dH₂O or until excess crystal violet is removed, and plaques are easily visualized.

• 11.

  Count the plaques at the dilution in which there are 10–100 plaques. Calculate titer in PFU/mL using the following formula: Titer (PFU/mL)=number of plaques counted×10^^{dilution counted}×5 (to get to mL because we added 200 μL of diluted sample)

Quantification of SARS-CoV-2 by focus-forming assay. A focus-forming assay is similar to a plaque assay in that it detects infectious virus in a sample. A “foci” describes the zone of cells that have become infected from a single infectious unit. These foci of cells express high amounts of viral antigen, which can be detected using a virus-specific antibody that is directly conjugated to a colorimetric readout (e.g. peroxidase) or through use of secondary antibodies (Fig. 3). This approach adds specificity to the assay, but also increases the number of processing steps post-infection. However, because the focus-forming assay captures infected foci before the cells die and develop into plaques, this assay typically requires shorter incubation times than the plaque assay. It also can be performed in 96-well plate format, which can increase throughput.

2.6. Materials needed

Vero E6 or Vero-furin cells

Vero cell culture medium (see Recipes)

Infection media (see Recipes)

Viral sample to be titered

96-well U-bottom plates

96-well flat-bottom plates

2X MEM+4% FBS (see Recipes)

2% methylcellulose (see Recipes)

4% PFA

Permeabilization (Perm) wash buffer (see Recipes)

Anti-SARS-CoV-2 antibody (and secondary antibody if primary is not directly conjugated)

Automated cell counter (e.g. CTL BioSpot)

• 1.

  Plate Vero E6 or Vero-furin cells in 96-well flat bottom plates at a cell density of 2.5×10⁴cells/well in a total volume of 100 μL/well.

• 2.

  Incubate cells overnight (12–18h) in humidified incubator at 37°C in 5% CO₂.

• 3.

  The next day, make 10-fold serial dilutions of viral sample to be titered in infection media in 96-well U-bottom plates. Change tips between dilutions and mix each row well before transferring to the next.

• 4.

  Remove existing cell culture media from cells. Transfer 100μL of the dilutions generated in step #3 to corresponding wells on cell plate starting with the most diluted so the same pipette tip can be used up the dilution series.

• 5.

  Incubate at 37°C for 1h.

• 6.

  Meanwhile, mix 2X MEM + 4% FBS with 2% methylcellulose in a 1:1 ratio. Warm in 37°C incubator while plates are incubating to decrease viscosity of the solution.

• 7.

  Add 100 μL/well MEM:methylcelluose overlay to each well.

• 8.

  Incubate at 37°C for 30h.

• 9.

  Remove methylcellulose:MEM overlays from each well.

• 10.

  Add 300μL of 4% PFA in PBS (see Recipes) to each well.

• 11.

  Incubate at room temperature for 20min.

• 12.

  Remove 4% PFA into appropriate waste container.

• 13.

  Wash cells with 300μL of Perm wash (see Recipes) six times to remove any remaining overlay and 4% PFA.

*Plates can now be removed safely from the BSL3 after obtaining Institutional Biosafety Committee approval.

• 14.

  Add 50μL primary antibody/well in Perm wash. Incubate at 4°C overnight with no rocking or 2hat room temperature with rocking.

*These conditions are optimized for using CR3022 (Yuan et al., 2020) at 1μg/mL.

• 15.

  Wash three times with 300 μL/well of PBS+Tween wash (see Recipes).

• 16.

  Add 50μL of secondary antibody/well in Perm wash.

*We use goat anti-human IgG-HRP at 1:500 (Sigma).

• 17.

  Incubate plates for 2hat room temperature with rocking or gentle agitation.

• 18.

  Wash 3x with 300 μL/well of PBS+Tween wash.

• 19.

  Add 50μL of KPL TrueBlue substrate (from Seracare Life Sciences Inc).

• 20.

  Incubate plates at room temperature with rocking until foci are fully developed and visible by eye (~15–30min).

• 21.

  Wash 3x with 300 μL/well of dH₂O.

• 22.

  Tap plate dry on a paper towel and image with CTL Immunospot plate reader.

Detection of SARS-CoV-2 antigen by flow cytometry. Detection of viral antigen in infected cells using flow-cytometry has a range of applications. In particular, multiplexing of viral staining with live/dead dyes and additional antigen stains can be used to screen infected cells for antibody binding or interrogate a range of biological variables.

2.7. Materials needed

Infected cells

Cell culture medium

96-well V-bottom plates

Dissociation reagent (e.g. Trypsin-EDTA or TrypLE)

FACS wash (see Recipes)

Anti-SARS-CoV-2 primary antibody

Secondary antibody (if primary is not directly conjugated)

• 1.

  If cells are adherent, dissociate SARS-CoV-2-infected cells into single-cell suspension using trypsin or EDTA-based dissociation agent.

• 2.

  Add cellular growth media and centrifuge in a swinging bucket rotor for 5minat 500×g.

Note: this must be performed in an aerosol-tight bucket with gasketed lid.

• 3.

  Open the gasketed centrifuge lid in the biosafety cabinet, remove samples, and pipette off supernatant. Resuspend cells in 4% PFA (final concentration) diluted in PBS and incubate for 10minat room temperature.

• 4.

  Centrifuge at 600×g for 3minat 4°C, remove supernatant, and resuspend cells in FACS wash.

*Cells can now be removed safely from the BSL3 after obtaining Institutional Biosafety Committee approval.

• 5.

  Count cells and add 3×10⁴–1×10⁶cells/well in a 96-well U-bottom plate for each sample to be tested.

*If desired, cells can be permeabilized using Perm wash (see Recipe below) and stained for intracellular antigen.

• 6.

  Centrifuge at 600×g for 3minat 4°C, remove supernatant, and resuspend cells with 50μL/well FACS wash containing primary antibody (e.g. containing 2μg/mL of CR3022). Incubate at 4°C for 45min–1h.

• 7.

  Wash twice with FACS wash by repeated centrifugation at 600×g for 3minat 4°C, removal of supernatant, and resuspension of pellet in FACS wash.

• 8.

  Upon completing the second spin, resuspend cells with FACS wash containing fluorophore-conjugated secondary antibody that recognizes the primary antibody (e.g., goat anti-human IgG Alexa 647at 1:1000 dilution) for 1hat 4°C.

• 9.

  Wash twice with FACS wash by repeated centrifugation at 600×g for 3minat 4°C, removal of supernatant, and resuspension of pellet in FACS wash. Resuspend after final spin in an appropriate volume for the flow cytometer you will use.

• 10.

  Analyze cells on a flow cytometer (Fig. 4).

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  Fig. 4

  SARS-CoV-2 infected cell flow cytometry plots. Indicated cell types were inoculated with SARS-CoV-2 at an MOI of 0.01PFU/cell. At each indicated timepoint post-infection, cells were collected and prepared for flow cytometry using CR3022 anti-S as the primary antibody followed by goat-anti-human IgG Alexa 647 as the secondary antibody.

SARS-CoV-2 outgrowth assay for validation of inactivation methods. To evaluate many aspects of COVID-19 biology, methods for inactivating SARS-CoV-2 infectivity are needed so that samples can be worked with safely outside of the BSL3. To test whether a specific method or treatment completely inactivates SARS-CoV-2, a virus outgrowth assay should be used. This type of assay is highly sensitive in that it allows for the outgrowth of as little as a single infectious unit. However, it is not quantitative and must be adapted to the application in question. Alternate agents and methods are benchmarked against “gold-standard” methods of inactivation of SARS-CoV-2 (Table 2). We describe methods that we have tested and validated, although before use, individual Institutional Biosafety Committees likely will need to review data before providing clearance.

2.8. Materials needed

Inactivation agent/method of choice

Vero E6 cells

Vero cell culture medium

2.9. Selecting an inactivation agent/method

Selecting the appropriate inactivation agent/method requires a detailed understanding of the project in question: specifically, the properties of the measurand (e.g., DNA, RNA, protein, and cells); the effect of the agent/method on the integrity of the measurand (e.g., fragmentation of DNA by formaldehyde or lysis of RNA by boiling); and the types of specimens that will be treated for inactivation (e.g., whole blood, plasma, cell culture media, cells, and tissues). No one reagent or method works for all applications, and ideally, the activity of each inactivation agent should be tested against each specimen type that will be used in the project. Validation of reagents/methods with a diverse array of applications may result in fewer hours spent performing validation of inactivation reagents.

2.10. Determination of the inactivation agent's cytotoxicity

Many commonly used chemical inactivation agents (e.g., chaotropic salts, detergents, and formaldehyde-based solutions) are toxic to cells (Fig. 5A). Because viruses require infection of a cell to replicate, this toxic effect of the inactivation agent must be diluted sufficiently after the sample has been treated to enable virus outgrowth.

• 1.

  (Day −1) Add 2×10⁴cells per well in a flat-bottom tissue-culture 96 well plate and incubate at 37°C overnight (12–18h).

• 2.

  (Day 0) In a separate U-bottom plate, add 200μL of the fully-reconstituted inactivation agent to well A1. Make serial 10-fold dilutions down the first column by transferring 18μL into 162μL. Make serial 3-fold dilutions across the plate using a multichannel pipette by transferring 60μL into 120μL; add only media to column 12.

This setup will create a dilution scheme in which the dilution that renders the inactivation agent non-toxic can be assessed in multiple rows.

• 3.

  Remove the supernatant from the plate containing Vero cells, and add 100 μL from each corresponding well in the plate containing diluted inactivation agent. Incubate cells at 37°C overnight.

• 4.

  (Day 1) Observe the cells under the microscope, noting wells with obvious cell death. Longer incubation times may be necessary. Mark wells in which toxicity is no longer obvious, and test the cells in these wells for viability and total cell number using trypan blue staining (or a variety of other live/dead counting methods). Use the media-only wells in column 12 for comparison. Wells that have the same viability and number of cells (±10%) should be used to calculate the required dilution factor.

2.11. Sample inactivation and virus outgrowth assay

To determine the ability of the agent/method to fully inactivate infectious virus, several high-titer specimens should be identified. If these are not available, then these specimens can be created by spiking specimens with SARS-CoV-2 virus stock (1:10) or infected cells. Ideally, specimens with the range of characteristics that will be encountered during the project (e.g., icteric, hemolyzed, and lipemic serum specimens) should be tested.

• 1.

  Prepare Vero E6 cells in a volume that enables the dilution of inactivation agent to a non-toxic level when >1μL of treated sample is added. The number of cells also should be such that they will reach approximately 50% confluency upon adhering.

For example, for diluting an agent 1:1000, plate 3-4×10⁴Vero E6 cells in 4mL and add 4 μL of the inactivated sample.

• 2.

  For the specimen(s) to be tested, split into two equal aliquots. Subject one to inactivation and the other to mock-inactivation (e.g., with addition of saline or medium instead of inactivation reagent). This should be performed at the temperature and for the duration of time that will be used for inactivation of experimental specimens.

• 3.

  Add the appropriate volume of inactivated (and mock-inactivated) sample to the Vero cells.

• 4.

  Incubate at 37°C and observe daily (Fig. 5B). Once obvious signs of CPE are observed, examine the cells and/or supernatant for infection using the flow cytometry (Figure.5C) and/or focus forming assay described above to confirm viral infection.

Conclusions. The emergence of SARS-CoV-2 and the resulting COVID-19 pandemic has strained biomedical resources throughout the world. Necessarily, pressure has been placed on the scientific community to deliver countermeasures for this continually evolving threat. Although new technologies are being applied to address this problem, classical virological methods, such as those presented here, remain important. Within a remarkably short period of time, the scientific community has built an infrastructure for studying SARS-CoV-2, especially given the biosafety concerns surrounding SARS-CoV-2 research. However, given the complex nature of COVID-19 pathophysiology, a critical need remains for developing new modalities for studying and combating this novel disease. Further optimization of assays will be required, and these will include a need to amplify high-titer virus stocks from low-passage patient isolates and develop new culture models to evaluate infectivity, host responses, and outcomes. New methods for SARS-CoV-2 inactivation will be developed, and these will require rigorous validation before wide-scale implementation. Issues regarding SARS-CoV-2 biosafety and biocontainment will continue to evolve as the pandemic progresses, and methods for safely working with and titrating SARS-CoV-2 will require further evaluation.

3.1. Vero cell culture medium

*For 1L:

Dulbecco's Modified Eagle Medium (high glucose) supplemented to contain:

10% heat-inactivated fetal bovine serum

1% Glutamax

10mM HEPES

100 U/mL penicillin/100 U/mL streptomycin

Sterile filter and store at 4°C.

*Vero-furin media is as stated above with the addition of 5μg/mL blasticidin.

3.2. MA104 culture medium

*For 1L:

M199 medium with Earle's salts supplemented to contain:

5% fetal bovine serum

10mM HEPES

100 U/mL penicillin/100 U/mL streptomycin

2.5μg/mL amphotericin B

3.3. Infection medium

*For 1L:

Dulbecco's Modified Eagle Medium (high glucose) supplemented to contain:

2% heat-inactivated fetal bovine serum

10mM HEPES

100U/mL penicillin/100U/mL streptomycin

3.4. 2X minimal essential medium+4% FBS

*For 1L:

200mL 10X MEM (Sigma #M0275)

20mL of 1M l-glutamine

20mL 1M HEPES

40mL heat-inactivated fetal bovine serum

4.2g sodium bicarbonate

20mL of 10,000 IU/ml penicillin +10,000μg/ml streptomycin

To volume with deionized distilled (Milli-Q) water

Sterile filter and store at 4°C.

3.5. 2% methylcellulose

Autoclave a 250mL glass bottle containing 2g carboxymethylcellulose powder (Sigma #M0512) and a stir bar.

Autoclave 100mL deionized distilled (Milli-Q) water.

When water is cool enough to handle, add to methylcellulose containing bottle.

Stir mixture overnight at 4°C and then store at 4°C until ready for use.

3.6. Crystal violet staining solution

*For 100mL:

50mg of crystal violet powder (Fisher #C581-100)

20mL of 100% methanol

80mL of deionized distilled (Milli-Q) water

Store at room temperature and mix well before each use.

3.7. Perm wash

*For 1L:

1g of saponin (Sigma, Cat. No: S7900)

1g of bovine serum albumin (Fraction V)

To volume with 1L phosphate buffered saline (without Ca or Mg)

Filter sterilize and store at 4°C until ready for use.

3.8. FACS wash

*For 1L:

100mL 10X phosphate-buffered saline

5mL 1M EDTA solution

50mL 5% heat-inactivated fetal bovine serum

10mL 5% sodium azide solution

835mL deionized distilled (Milli-Q) water

Filter sterilize and store at 4°C until ready for use.

3.9. PBS + Tween wash

1 L 10X PBS

9 L ddH₂O

50 mL 10% Tween-20

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