Aug 23, 2024

Infecting Cells with SeV or RSV in A549 or LLCMK2 cells V.1

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gpzeojlzp/v1
  • 1Washington University
Carolina Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gpzeojlzp/v1
Protocol Citation: Carolina Lopez 2024. Infecting Cells with SeV or RSV in A549 or LLCMK2 cells. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzeojlzp/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: July 26, 2024
Last Modified: August 23, 2024
Protocol  Integer ID: 104183
Keywords: infecting cells with sev, llcmk2 cells infection of sev, infecting cell, llcmk2 cells infection, sev, rsv in a549, rsv, cell
Abstract
Infection of SeV or RSV in A549 or LLCMK2 cells
Materials
INFECTION MEDIUM – SeV: Filter through 0.2 µm filter
ComponentAmountConc. Supp.Product information
DMEM500 mLGibco Cat. # 11965092 (#11965118-cs)
Pen/Strep5.0 mL100 U/mL Pen and 100 µg/mL StrepGibco Cat # 15140-122 – 10,000 U/mL
BSA 35%5.0 mL0.35%Sigma Aldrich Cat #A7979 – 35%
Sodium Bicarbonate (NaHCO3) 7.5%12 mL0.18%Gibco Cat #25080094 – 7.5%

TRYPSIN – TPCK (Worthington Biochemicals code: TRLVMF cat.no. LS004454)

INFECTION MEDIUM – RSV: Filter through 0.2 µm filter
ComponentAmountConc. Supp.Product information
DMEM500 mLGibco Cat. # 11965092 (#11965118-cs)
Gentamicin500 µL 50µg/mLGibco Cat #15750060 (#15750078-pk) – 50 mg/mL
Sodium Pyruvate 5.0 mL1mMCorning Cat #25-000-Cl – 100mM
L-Glutamine 5.5 mL 2 mMSigma Aldrich Cat #G7513 – 200mM
FBS10 mL2%


Infecting Cells with SeV or RSV in A549 or LLCMK2 cells
Infecting cells with SeV or RSV in A549 or LLCMK2 cells 1. Prepare a virus dilution in infection media with the correct virus-specific media (see materials section for media composition).
  • Calculate the volume of virus needed (X) using the MOI formula:
(MOI*cell number)/Virus titer = X
  • The amount of infection media used to prepare the virus will depend on the size of the well or plate used - it is best to use the minimal volume needed to cover the well/plate to help ensure virus particle attachment to cells
2. Remove TCM 3. Wash cells twice with 1X PBS 4. Add XµL of virus suspended in infection media to each well (calculated in step 1)
5. Incubate the plate for 1 hr @ 37°C, rocking the plate/flask every 15 minutes
6. Remove the media
7. Wash twice with 1X PBS 8. Add infection media to each well/flask
  • For SeV infection: add SeV-Infection media, for SeV infection in LLCMK2 cells: add TRYPSIN –TPCK to the infection media for a final concentration of 2µg/mL TRYPSIN –TPCK
  • For RSV infection: add RSV-Infection media (2%FBS)
9. Place the plate/flask back in a 37ºC, 5% CO2 incubator