Oct 02, 2025

Version 1

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# RSV Expansion and Titration V.1

DOI

https://dx.doi.org/10.17504/protocols.io.5qpvok39xl4o/v1

Carolina Lopez¹

¹Washington University

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Carolina Lopez

Washington University

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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvok39xl4o/v1

**Protocol Citation:** Carolina Lopez 2025. RSV Expansion and Titration. protocols.io

https://dx.doi.org/10.17504/protocols.io.5qpvok39xl4o/v1Version created by **Sydney Faber**

**License:** This is an open access protocol distributed under the terms of the **Creative Commons Attribution License**, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

**Protocol status:** Working

We use this protocol and it's working

Created: October 01, 2025
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**Last Modified:** December 03, 2025

**Protocol Integer ID:** 228799

**Keywords:** rsv expansion, titration protocol, protocol, expansion

Abstract

Protocol to expand and titrate RSV
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## Materials

Trypsin-EDTA, 0.05% Thermo Fisher Scientific Cat #25300054

PBS Gibco Cat #20012027

**TISSUE CULTURE MEDIUM (TCM):** Filter through 0.2 µm filter

<table><thead><tr><td>Component</td><td>Amount</td><td>Conc. Supp.</td><td>Product information</td></tr></thead><tbody><tr><td>DMEM</td><td>500 mL</td><td></td><td>Gibco Cat. # 11965092 (#11965118-cs)</td></tr><tr><td>Gentamicin</td><td>500 µL</td><td>50µg/mL</td><td>Gibco Cat #15750060 (#15750078-pk) - 50 mg/mL</td></tr><tr><td>Sodium Pyruvate</td><td>5.0 mL</td><td>1mM</td><td>Corning Cat #25-000-Cl - 100mM</td></tr><tr><td>L-Glutamine</td><td>5.5 mL</td><td>2 mM</td><td>Sigma Aldrich Cat #G7513 - 200mM</td></tr><tr><td>FBS</td><td>50 mL</td><td>10%</td><td></td></tr></tbody></table>

**INFECTION MEDIUM - RSV:** Filter through 0.2 µm filter

<table><thead><tr><td>Component</td><td>Amount</td><td>Conc. Supp.</td><td>Product information</td></tr></thead><tbody><tr><td>DMEM</td><td>500 mL</td><td></td><td>Gibco Cat. # 11965092 (#11965118-cs)</td></tr><tr><td>Gentamicin</td><td>500 µL</td><td>50µg/mL</td><td>Gibco Cat #15750060 (#15750078-pk) - 50 mg/mL</td></tr><tr><td>Sodium Pyruvate</td><td>5.0 mL</td><td>1mM</td><td>Corning Cat #25-000-Cl - 100mM</td></tr><tr><td>L-Glutamine</td><td>5.5 mL</td><td>2 mM</td><td>Sigma Aldrich Cat #G7513 - 200mM</td></tr><tr><td>FBS</td><td>10 mL</td><td>2%</td><td></td></tr></tbody></table>

Disposable cell scraper Fisher Scientific Cat #08-100-241

Ultra-Clear™ centrifuge tubes, 25 × 89 mm Beckman Coulter Cat #344058

Hanks' balanced salt solution (HBSS) Gibco Cat #14-025-092

Crystal violet Sigma Cat #C0775

## CRYSTAL VIOLET SOLUTION

* **STOCK:** 1% Crystal Violet powder in 20% $C_2H_5OH$ solution
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To make 100mL STOCK:

1g Crystal Violet

20mL absolute C₂H₅OH (ethanol)

80mL water

## Working Dilution:

40mL STOCK

80mL CH₃OH (methanol)

180mL dd H₂O

## Troubleshooting
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## Cell Preparation

1. Seed 1×10⁷ HEp2 cells per T225 flask the night before
   Typically, seed 1-2 flasks for a seed stock and 4-8 flasks for a working stock

## RSV Infection

2. Wash cells once with PBS

3. For LD stock: Infect HEp2 cells with RSV-LD at an MOI of 0.01
   For HD stock: Infect HEp2 cells with RSV-LD at an MOI of 4

**Note**

LD infection: stock containing low levels of non-standard viral genomes

HD infection: stock containing high levels of non-standard viral genomes

Dilute virus in RSV infection media

Add ⬇ 4-5 mL of diluted virus to each T225 flask

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4. Incubate at ⬇ 37 °C for ⏻ 01:00:00 rocking flasks every ⏻ 00:15:00 to maintain an even virus distribution and avoid the cells drying out

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1h

5. After ⏻ 01:00:00 incubation, add ⬇ 25 mL RSV infection media to each flask

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6. Incubate at ⬇ 37 °C

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## Harvest

7. Check the progress of infection
   - LD - harvest at 4-5dpi, may or may not see cell death
   - HD - harvest at 2dpi, or until half the cells are dead
   If the virus has fluorescence, use that to ensure productive infection is visible before harvesting

8. Scrape the cells and collect them together with the culture supernatant
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9 Centrifuge at 1180 rpm for 00:05:00 at 4 °C and collect the supernatant into fresh tubes and place on ice 5m

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10 Add ⬇ 2-3 mL of supernatant back to the cell debris. Keep the remaining supernatant on ice

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11 Freeze (in a mixture of dry ice+ethanol) / thaw (in 37 °C water bath) the cell debris 3 times, vortex in between each thaw and freeze

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Note

This step is to help aid release the virions from the cell surface and help increase the titer

12 Centrifuge cell debris at 1180 rpm for 00:05:00 and only take the supernatants and combine with the supernatant on ice. Discard the debris. 5m

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13 For seed stocks (P1), you can stop here and aliquot the virus supernatant and titer. Once the virus is aliquoted, snap freeze in dry ice + ethanol before storing in -80 °C
For working stock (P2), it's recommended to follow steps 14-17 to achieve a higher titer stock

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14 Ultra-centrifuge the supernatants ⚙️ 59000 x g, 4°C, 02:30:00 using a SW 32Ti Rotor and Ultra-Clear™ 25×89 mm centrifuge tubes to concentrate the working virus stock 2h 30m

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15 Discard the supernatant and lightly wash the pellet with ⬇ 1 mL HBSS twice without disturbing the pellet

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16 Resuspend pellet in RSV infection media ⬇ 200-800 µL per T225 flask
*Resuspension volume can be adjusted based on desired titer and may depend on the strain of RSV

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17 Aliquot (100 µL/vial) and snap freeze in dry ice + ethanol before storing in 37 °C

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TCID50 - Titration of RSV

18 Prepare 96-well plates with HEp2 cells (2×10⁶ cells/10mL of TCM). Seed ⬇ 100 µL per well the night before. Cells should be 80-90% confluent at the time of infection

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19 Prepare a dilution plate with serial 1/10 dilutions of the virus in triplicate. Remember to include a positive control and a negative (media alone). Change tips between each dilution

20 Remove media from cells and wash once with PBS

21 Transfer ⌭ 25 µL of virus dilution/well. Start with the lowest dilution - changing tips is not necessary

22 Incubate at ⌭ 37 °C for 🕒 01:00:00 rocking flasks every 🕒 00:15:00 to maintain an even virus distribution and avoid the cells drying out

1h

23 Add ⌭ 175 µL of RSV infection media to each well

24 Incubate for 4-5 days and look at CPE or perform the following crystal violet staining

25 Discard media into a container containing bleach

26 Add ⌭ 50 µL of crystal violet working solution to each well. Be careful not to touch the bottom of the wells

27 Incubate at ⌭ Room temperature for 🕒 00:20:00 - 🕒 00:30:00

30m

28 Discard crystal violet and wash the plate by submerging it upside down in water several times to eliminate the excess crystal violet

29 Let plate air dry

30 Determine the endpoint dilution by reading the CPE in the monolayer

31 Score the titer by determining the last dilution with positive CPE. Score the number of positive wells for that last dilution (number of positive out of three). From this score,
calculate the TCID50/25µL using the following formula, where "x" corresponds to the last dilution with positive CPE:

$$ "+++" 10^x \text{ TCID}_{50} = 10^{x+0.7}/25 \text{ µL} $$

$$ "+--" 10^x \text{ TCID}_{50} = 10^{x+0.4}/25 \text{ µL} $$

$$ +--" 10^x \text{ TCID}_{50} = 10^{x-0.1}/25 \text{ µL} $$

<figure><img src="image_26.png" /><figcaption>Figure 3 from Preparation of respiratory syncytial virus stocks and purification of defective viral particles. Bio-protocol 6(10): e1820. http://www.bio-protocol.org/e1820.
**Crystal violet staining of TCID₅₀.** The titers from three different viruses were determined by TCID₅₀ as illustrated above. 10⁻⁴ and 10⁻⁵ were the last dilutions with positive CPE for designated virus samples, which were the "x". "+" stands for the positive CPE observed. "-" indicates no CPE observed. Based on the equation above, calculate the final titer of each virus as shown at the bottom of this figure.</figcaption></figure>

Note

For working virus stocks, we typically do 3 independent repeats of the TCID50, then calculate the average for the working virus stock titer that will be used in future experiments
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Protocol references

Lopez Lab

For details about this protocol, please refer to Preparation of respiratory syncytial virus stocks and purification of defective viral particles. Bio-protocol 6(10): e1820. http://www.bio-protocol.org/e1820.