Dec 03, 2025
RSV Expansion, Purification, and Titration
Forked from RSV Expansion and Titration
- 1Washington University
Protocol Citation: Carolina Lopez 2025. RSV Expansion, Purification, and Titration. protocols.io https://dx.doi.org/10.17504/protocols.io.eq2ly4odqlx9/v1
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: December 01, 2025
Last Modified: December 03, 2025
Protocol Integer ID: 233895
Keywords: rsv expansion, titration protocol, protocol, rsv, purification
Abstract
Protocol to expand and titrate RSV
Materials
Trypsin-EDTA, 0.05% Thermo Fisher Scientific Cat #25300054
PBS Gibco Cat #20012027
TISSUE CULTURE MEDIUM (TCM): Filter through 0.2 µm filter
| Component | Amount | Conc. Supp. | Product information | |
| DMEM | 500 mL | Gibco Cat. # 11965092 (#11965118-cs) | ||
| Gentamicin | 500 µL | 50µg/mL | Gibco Cat #15750060 (#15750078-pk) – 50 mg/mL | |
| Sodium Pyruvate | 5.0 mL | 1mM | Corning Cat #25-000-Cl – 100mM | |
| L-Glutamine | 5.5 mL | 2 mM | Sigma Aldrich Cat #G7513 – 200mM | |
| FBS | 50 mL | 10% |
INFECTION MEDIUM – RSV: Filter through 0.2 µm filter
| Component | Amount | Conc. Supp. | Product information | |
| DMEM | 500 mL | Gibco Cat. # 11965092 (#11965118-cs) | ||
| Gentamicin | 500 µL | 50µg/mL | Gibco Cat #15750060 (#15750078-pk) – 50 mg/mL | |
| Sodium Pyruvate | 5.0 mL | 1mM | Corning Cat #25-000-Cl – 100mM | |
| L-Glutamine | 5.5 mL | 2 mM | Sigma Aldrich Cat #G7513 – 200mM | |
| FBS | 10 mL | 2% |
Disposable cell scraper Fisher Scientific Cat #08-100-241
Ethylenediaminetetraacetic acid disodium salt solution (EDTA) pH 8.0, 0.5M Sigma Aldrich Cat #03690
D-Sucrose (Molecular Biology) Fisher Scientific Cat #BP220-1
Ultra-Clear™ centrifuge tubes, 25 x 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% C2H5OH solution
To make 100mL STOCK:
1g Crystal Violet
20mL absolute C2H5OH (ethanol)
80mL water
- Working Dilution:
40mL STOCK
80mL CH3OH (methanol)
180mL dd H2O
Cell Preparation
Seed 1x107 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
Wash cells once with PBS
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
Incubate at 37 °C , 5% CO2 for 01:00:00 rocking flasks every 00:15:00 to maintain an even virus distribution and avoid the cells drying out
1h
After 01:00:00 incubation, add 25 mL RSV infection media to each flask
Incubate at 37 °C , 5% CO2
Harvest + Purification
2h 10m
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
Prepare PNE buffer:
50 mL PBS supplemented with 200 µL 0.5M EDTA
(adjust volumes depending on how many flasks are being harvested)
Prepare 33% sucrose:
16.5 g sucrose dissolved in 50 mL PNE buffer
(adjust volumes depending on how many flasks are being harvested)
Scrape the cells and collect them together with the culture supernatant
Centrifuge at 1180 rpm for 00:05:00 at 4 °C and collect the supernatant into fresh tubes and place on ice
5m
Add 2-3 mL of supernatant back to the cell debris. Keep the remaining supernatant on ice
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
Note
This step is to help aid release the virions from the cell surface and help increase the titer
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
Dilute the virus mixture by adding additional RSV infection media - up to 1:1
Note
A 1:1 mix is a suggestion, but not required. The more dilute the stock is, the better it will be able to separate through the sucrose cushion. However, dilution volume can be determined by calculating the total volume from all flasks and how many tubes/rounds of ultra-centrifugation will be done. For example, one round of ultra-centrifugation using all 6 tube holders will allow for a maximum of 180 mL virus mixture. Therefore, multiple rounds of ultra-centrifugation will be required if harvesting 8 flasks.
Add 8 mL of the 33% sucrose solution to each Ultra-Clear™ 25x89 mm centrifuge tube to prepare the sucrose cushion
Add 30 mL of virus mixture to each tube. Slowly add by pipetting on the side of the tube to try to avoid any mixing with the sucrose layer. Place any remaining virus mixture on ice.
Ultra-centrifuge the supernatants in the Ultra-Clear™ 25x89 mm centrifuge tubes at 28000 rpm, 4°C, 02:00:00 using a SW 32Ti Rotor and Optima XPN-100 Ultracentrifuge to concentrate the working virus stock
2h
Discard the supernatant and lightly wash the pellet with 1 mL HBSS twice without disturbing the pellet
Note
If the pellet is very small or loose, the washing can be omitted to avoid losing particles. However, attempt to remove all liquid as best as possible before resuspending the pellet.
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
*Can resuspend in PBS instead of infection media if needed
Repeat steps 16-20 if multiple rounds of ultracentrifugation are required.
Aliquot (100 µL/vial) and snap freeze in dry ice + ethanol before storing in -80 °C
TCID50 - Titration of RSV
Prepare 96-well plates with HEp2 cells (2x106 cells/10mL of TCM). Seed 100 µL per well the night before. Cells should be 80-90% confluent at the time of infection
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
Remove media from cells and wash once with PBS
Transfer 25 µL of virus dilution/well. Start with the lowest dilution - changing tips is not necessary
Incubate at 37 °C , 5% CO2 for 01:00:00 rocking flasks every 00:15:00 to maintain an even virus distribution and avoid the cells drying out
1h
Add 175 µL of RSV infection media to each well
Incubate for 4-5 days and look at CPE or perform the following crystal violet staining
Discard media into a container containing bleach
Add 50 µL of crystal violet working solution to each well. Be careful not to touch the bottom of the wells
Incubate at Room temperature for 00:20:00 - 00:30:00
30m
Discard crystal violet and wash the plate by submerging it upside down in water several times to eliminate the excess crystal violet
Let plate air dry
Determine the endpoint dilution by reading the CPE in the monolayer
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:
"+++" 10x TCID50 = 10X+0.7/25 µL
"++-" 10x TCID50 = 10X+0.4/25 µL
"+--" 10x TCID50 = 10X-0.1/25 µL
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.
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
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.