Dec 11, 2017
Version 2
Fluorescent Focus Unit Assay using LICOR Imaging System V.2
- 1Emory University
External link: https://doi.org/10.1371/journal.ppat.1006768
Protocol Citation: Bernardo Mainou 2017. Fluorescent Focus Unit Assay using LICOR Imaging System. protocols.io https://dx.doi.org/10.17504/protocols.io.k8fcztn
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 11, 2017
Last Modified: March 15, 2018
Protocol Integer ID: 9191
Keywords: Reovirus, infectivity, FFU, fluorescent focus unit
Abstract
Indirect immunofluorescence infectivity assay for reovirus using LICOR Imaging System
Attachments
Licor FFU.docx
98KB
Guidelines
This assay is standardized to for 96-well plates using reovirus polyclonal antiserum. New batches of polyclonal antiserum or monoclonal antibodies should be tested to determine optimal dilution.
Before start
Plates and reagents:
Corning Black with clear bottom TC treated 96 well plates (Corning #3904)
DRAQ5 #4084 from Cell Signaling
Sapphire 700 #928-40022 from LICOR
Antibody 800 from LICOR (Life Technologies ones don’t work for this)
Remove media from wells.
Wash 1x with Phosphate Buffered Saline (PBS).
Add 100 uL ice-cold methanol per well.
Store plate at -20°C for at least 30 min (can remain at -20°C for several weeks).
Remove methanol, allow plate to come to room temperature, and excess ethanol to evaporate.
Wash wells with 150 ul PBS
Add 150 uL Dulbecco’s PBS (DPBS) with 0.5% Tween-20 (DPBS-T) to each well.
Remove immediately.
Add 50 uL rabbit anti-reovirus polyclonal antiserum (1:1000 dilution) in DPBS with 1% BSA (DPBS-BSA) to experimental wells.
Add 50 uL DPBS-BSA with no antibody to background control.
Incubate for 1 h at 37°C.
Remove DPBS-BSA.
Wash 3x with DPBS-T for 5 min each wash while shaking.
Add 50 uL of DPBS-BSA to all wells.
Incubate 1 h at 37°C.
Wash plates 3x with DPBS-T for 5 min each wash.
Prepare cell staining solution in DPBS-BSA:
Secondary antibody (e.g. Goat Anti Rabbit LICOR IRDye 800CW) - 1:1000
Draq5 - 1:10,000
Sapphire700 - 1:1000
Note: we have found that we get less background with the LICOR IRDye 800CW secondary antibody than with Goat Anti Rabbit Alexa 790)
Add 50 uL cell staining solution to all wells that were treated with primary.
Add 50 uL of DPBS-BSA with secondary antibody (1:1000) only to background control wells.
Remove solution.
Incubate 1 h at 37°C.
Wash 3x with 150 uL DPBS-T.
Add 50 uL of water.
Scan plates on LICOR Odyssey Imaging System.
a. Focus offset = 3.0 (depends on the plates)
b. 700nm Intensity = 6.5
c. 800nm Intensity = 7.5
Plates and reagents:
Corning Black with clear bottom TC treated 96 well plates (Corning #3904)
DRAQ5 #4084 from Cell Signaling
Sapphire 700 #928-40022 from LICORAntibody 800 from LICOR (Life Technologies secondary antibodies give extra background and should be avoided)