Feb 01, 2025

Public workspaceSeV fluorescent reporter virus titration by TCID50

Journal of Virology
DOI
https://dx.doi.org/10.17504/protocols.io.ewov1d73yvr2/v1
  • Carolina Lopez1
  • 1Washington University
Carolina Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.ewov1d73yvr2/v1
External link: https://doi.org/10.1128/jvi.00894-25
Protocol Citation: Carolina Lopez 2025. SeV fluorescent reporter virus titration by TCID50. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d73yvr2/v1
Manuscript citation:
Pye SE, Achouri E, Yang Y, Musa A, López CB Validation of diverse and previously untraceable Sendai virus copyback viral genomes by direct RNA sequencing. Journal of Virology 99(8). doi: 10.1128/jvi.00894-25
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: January 14, 2025
Last Modified: February 01, 2025
Protocol Integer ID: 118345
Keywords: sev fluorescent reporter virus titration by tcid50 protocol, sev fluorescent reporter virus titration, tcid50 protocol, virus, rsev
Abstract
Protocol to titer rSeV carrying a fluorescent reporter
Materials
  1. Cultured LLCMK2 cells (ATCC, #CCL-7)
  2. Tissue culture media and Infection media (See media prepare protocol)
  3. 0.05% Trypsin (Thermo, #25300054)
  4. PBS (Phosphate-buffered saline)
  5. Cell counter and Trypan blue
  6. 96-well plates
  7. Pipettes, 1.5 ml tubes
  8. TPCK-Trypsin (for Sendai virus infection, see SeV infection protocol)
Troubleshooting
Seed cells to 96-well plate
  1. Check LLCMK2 cells to ensure they are in good condition, approximately 90% confluent.
  2. Wash cells twice with PBS.
  3. Add Amount3 mL 0.05% trypsin-EDTA to the T75 flask, incubate at room temparture for 3-4 minutes.
  4. Add Amount7 mL TCM to the flask to stop trypsinization and collect cells.
  5. Mix Amount10 µL cells and Amount10 µL Trypan blue, then add Amount10 µL mixture to a cell counter slide and count the cells.
  6. Plate 20,000 cells in Amount100 µL of TCM media to each well. 3 rows of a 96-well plate are need for one test.
  7. Incubate plates at Temperature37 °C overnight.

Notes:
  • Usually, LLCMK2 cells are used for SeV titration since most subsequent experiments are done with LLCMK2. However, if you plan to infect A549 cells or other cell lines with the virus stocks, it is recommended to do the titration on the same cell line you intend to use.
  • The seeding number for LLCMK2 and A549 cells is similar, usually around 20,000 cells per well. This results in 95–100% confluence overnight, though this can vary depending on the condition of the cells. In my experiments, seeding numbers between 12,000 and 20,000 cells per well have been successfully used.

Titration
  1. Prepare infection media: Add TPCK-trypsin to the infection media at a final concentration of 2 µg/mL. Prepare 30 mL of the media for each plate, ensuring sufficient volume for dilutions and replacing the TCM from the plate. (This step is for LLCMK2 cells, if you are using A549 cells, TPCK-trypsin is not needed).
  2. Use the prepared media to do a 10-fold serial dilution from 1:10¹ to 1:10⁸: Prepare 8 tubes. Add 450 µL of prepared media to each tube. Add 50 µL of virus to the first tube, vortex to mix thoroughly, then transfer 50 µL to the second tube. Repeat this process sequentially until the 8th tube.
  3. Check the cells under a microscope to confirm they are healthy and evenly distributed.
  4. Remove the TCM from the plate using a multichannel pipette.
  5. Wash the cells by adding 200 µL of PBS to each well, then discard the PBS.
  6. Add 100 µL of the prepared infection media (containing TPCK-trypsin) to each well.
  7. Add 100 µL of the diluted virus to the cells, with each dilution to three wells (e.g., columns A-C as below).
  8. Incubate the plate at 37 °C for 3–5 days.
ABCDEFGHIJKL
Stock1 1:10^1Stock1 1:10^1Stock1 1:10^1Stock2 1:10^1Stock2 1:10^1Stock2 1:10^1...
Stock1 1:10^2Stock1 1:10^2Stock1 1:10^2Stock2 1:10^2Stock2 1:10^2Stock2 1:10^2
Stock1 1:10^3Stock1 1:10^3Stock1 1:10^3Stock2 1:10^3Stock2 1:10^3Stock2 1:10^3
Stock1 1:10^4Stock1 1:10^4Stock1 1:10^4Stock2 1:10^4Stock2 1:10^4Stock2 1:10^4
Stock1 1:10^5Stock1 1:10^5Stock1 1:10^5Stock2 1:10^5Stock2 1:10^5Stock2 1:10^5
Stock1 1:10^6Stock1 1:10^6Stock1 1:10^6Stock2 1:10^6Stock2 1:10^6Stock2 1:10^6
Stock1 1:10^7Stock1 1:10^7Stock1 1:10^7Stock2 1:10^7Stock2 1:10^7Stock2 1:10^7
Stock1 1:10^8Stock1 1:10^8Stock1 1:10^8Stock2 1:10^8Stock2 1:10^8Stock2 1:10^8

Read titration results
Read the results at 4dpi as below:

ABC
Positive wells of last row with signalDilution of last row with signalTCID50 / 100uL
+ + +10-X10^X + 0.7
+ + -10-X10^X + 0.4
+ - - 10-X10^X - 0.1
X = row in which last signal occurs
+ = Fluorescent signal positive

Notes:
  • The TCID50 result is reported per 100 µL. To convert it to per mL, plus 1 to the X. For example, a TCID50 of 107.7/100 µL is equivalent to 108.7 TCID50/mL.
  • For rSeV-C-eGFP, the TCID50 results remain consistent from 3 dpi to 5 dpi. So, the results can be read on any of these days.