Sep 13, 2022

Public workspaceGFP-VSV Infection V.1

This protocol is a draft, published without a DOI.
  • Christopher Rousso1,
  • Alison Macdonald1
  • 1UOttawa
Kelsey Grimes
Icon indicating open access to content
QR code linking to this content
Protocol Citation: Christopher Rousso, Alison Macdonald 2022. GFP-VSV Infection. protocols.io https://protocols.io/view/gfp-vsv-infection-cgcitsue
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: September 08, 2022
Last Modified: September 13, 2022
Protocol Integer ID: 69738
Abstract
GFP-VSV Infection
Materials
-Aliquot of GFP-VSV virus (from -80°C)
-Serological pipets
-1.5 mL Eppendorf tubes
-Serum free DMEM media (high glucose) [depending on cell type]
-DMEM media (high glucose + 10% FBS) [depending on cell type]
-Ice bucket
If infecting transfected cells, infect them 24 hours after transfection.
Remove cells from 37 °C incubator.
Replace media with fresh media (with FBS) prior to infection with GFP-VSV.
Thaw VSV-GFP stock aliquot on ice
Dilute VSV-GFP in serum-free culture media. Perform the necessary calculations to determine the dilution that is required to obtain the desired MOI (multiplicity of infection).
Note: MOI is the number of viruses/number of cells. For example, a MOI of 1 = 500,000 PFU (plaque forming units) of virus per 500,000 cells.
Note: We typically start with a dilution range of MOI = 0.01, 0.1, 1 for optimization
Infect cells by dropping the diluted virus directly into the wells making sure to disperse the virus around the well.
Gently swirl the plate to help disperse the virus.
Leave the plate for approximately 24 hours in a 37°C CO2 incubator. Then proceed with endpoint analysis.
Appendix - Sample Calculation
Appendix - Sample Calculation
Sample calculation:
(MO1 = 1; Virus titre = 8.4x108 virus/mL ; 500,000 cells/well;)
Step 1: calculate amount of PFU per well



Step 2: calculate how much to dilute the viral stock
C1V1=C2V2
Where V1 = volume/well you have
C1 = virus/mL calculated in step 1
C2 = virus titre (stock concentration)
V2 = x (unknown volume to be calculated)
You will add x mL of your virus stock per well. This volume will be low, and thus must be diluted in media for easier pipetting.