Guide: RRV Kit Readout on the Cytation

Summary

The protocols we provide for Ready Reporter Virus (RRV) kits are based on flow cytometric readout; however, RRV neutralization assays are also compatible with high-content imagers, such as the Agilent BioTek Cytation. This guide provides our recommendations on instrument specifications, assay setup, imaging acquisition, and analysis for the Cytation.

This guide provides information on:
  • LED/filter cube sets that have been tested with the RRV kit.
  • Laboratory consumables and assay setup are adapted to remove agitation of cells.
  • Recommendations on image analysis for infected cell counting.

High-content imagers offer a rapid and powerful means of reading out same-day RRV neutralization assays.

This guide is intended for use with RRV kits that use HEK-293T-derived target cells, listed below.

Introduction

This guide is for users of the Ready Reporter Virus (RRV) kit who have experience with the Agilent BioTek Cytation 5 automated microscopy platform. It may be used alongside our standard RRV infectivity and neutralization protocols for readout by fluorescence microscopy. With appropriate adaptation, RRV kit results are highly comparable between flow cytometric and imaging readouts.

Neutralization curves for flow cytometry- and image-based readouts show consistent NT50s, 0.33 to 0.32 nanograms per microliter, respectively.

Comparison of Flow Cytometry- and Imaging-Based Readout. SARS-CoV-2 D614G RRVs were incubated with neutralizing antibody (IM-CoV-2G06) or negative control antibody for 1 hour prior to infecting HEK-ACE2 Target Cells for 3 hours. Infected Target Cells were measured by flow cytometry (left) or imaging on the Cytation 5 (right). NT50 values were determined from lines of best fit to neutralization data.

RRV Kit Overview

Ready Reporter Virus (RRV) kits use a pseudovirus system that models authentic viral entry in the absence of viral genetic material. Each kit includes non-replicative, pseudotyped reporter virus particles that display relevant viral envelope proteins, along with permissive target cells pre-loaded with reporter dye. When the viral particles fuse with the cell membrane, they deliver an enzyme reporter. The enzyme triggers a change in the fluorescent reporter substrate inside the infected cell. This fluorescent signal allows you to quantify viral infectivity or neutralization by microscopy.

Upon excitation at 409 nm, target cells with no RRV entry (uncleaved dye) emit at 520 nm, while target cells with RRV entry (cleaved dye) emit at 447 nm.

Excitation and emission spectra in an uninfected (green box, top) or infected (blue box, bottom) Target Cell. Representative images of overlaid 520 nm and 447 nm fluorescence.

Assay Plate Setup

Infectivity or neutralization assay setup for imaging readout largely follows the protocols for readout by flow cytometry, with adaptations listed in the table below. Recommended serial dilutions for neutralizing samples, RRV particle input, and Target Cell seeding density do not need to be adapted unless improvements are empirically determined.

Adaptations to assay plate setup

Protocol Step Protocol Component Adaptation for Imaging Readout
Plastics 96-well U-bottom assay plate 96-well Nunc optical-bottom plate
Neutralization Setup Plate shaking or mixing at each hour during 3 hour incubation Do not shake or mix plate.

Prior to imaging, spin down plates at 1500 rpm for 2 minutes.

Data Collection by Imaging

As during assay setup, plate shaking should not be enabled for readout by microscopy. Viable Target Cells fluoresce at 520 nm. Infected Target Cells fluoresce at both 520 nm and 447 nm. Therefore, acquiring and analyzing infection is performed with the Em. 409 nm/Ex. 447 nm channel. This guide describes acquisition and analysis using readily available catalog parts developed for mTagBFP imaging; custom LED and filter cube sets may further minimize background signal.

One hour after RRV particle infection, target cells show fluorescence mainly at 520 nm with some signal at 447 nm. Four hours post-infection, cells show strong fluorescence at both 520 nm and 447 nm, representing uncleaved and cleaved dye, respectively.

Representative images of fluorescence in infectivity assay. SARS-CoV-2 D614G RRV infection of HEK-ACE2 Target Cells (catalog #RRK-702). Images of uncleaved dye (Em. 520 nm), cleaved dye (Em. 447 nm), and merged channels. Images were taken at 1 hour post-infection (top row) and at 4 hours post-infection (bottom row).

Recommended Filter Cube Sets

Note on fixing samples: Fixation is a common step in sample preparation prior to microscopy. Fixation of RRV samples is not recommended prior to imaging because it causes reduced signal.

Note on DAPI staining: DAPI staining is a common step in sample preparation prior to microscopy to observe nuclear morphology, count total cells, and guide autofocus on high-content imagers. DAPI staining is not compatible with the fluorescent reporter in the RRV kit. The GFP channel readily detects all Target Cells and can be used for total cell counts or to guide autofocus.

Channel Name Excitation Emission Reporter
mTagBFP 409 nm
(1225009 | Agilent)		 447 nm
(1225115 | Agilent)		 Infected Target Cell
GFP 409 nm
(1225001 | Agilent)		 520 nm
(1225101 | Agilent)		 Target Cell

Acquisition and Analysis with the Cytation 5 and Gen5 Software

Image Acquisition: Principles and Suggested Settings

Gen5 Menu Gen5 Setting Suggested Value
Procedure Plate Type Nunc 96 well optical bottom plate
Procedure Actions Image
Imaging Step-Inverted Imager Magnification 4X
Imaging Step-Inverted Imager Channels (1) GFP, (2) TagBFP
Focus options... For Channel 1: Auto

For Channel 2: Focal height from first channel (+ offset): 0
Experiment Load Plate Use Lid
Image Analysis: Principles and Suggested Settings

Gen5 Menu Gen5 Setting Suggested Value
Data Reduction Image Analysis Cell Counting
Cell Counting Channel TagBFP
Cell Counting Threshold Value: determine for your instrument using Line Profile

Background: Dark
✓Split touching objects
Cell Counting Object selection Min. object size: 12 µm
Max. object size: 20 µm
Cell Counting Advanced detection options... ✓Background flattening
Rolling ball diameter: 20 µm
Image smoothing strength: 2
Threshold on background using Line Profile, split touching objects, and object selection set to 12-20 microns.

Representative Images showing examples of Gen5 software features for setting up Cell Count Analysis. Line Profile may be used to determine appropriate background and thresholding settings (left). Electing to split touching objects enables more accurate cell counting (middle). Selecting objects or Regions of Interest based on a size between 12-20 µm eliminates most debris in the field (right).

Frequently Asked Questions