Sample Preparation for Luciferase-Based Viral Quantification

Abstract

Source: Garcia, V., et al. High-throughput Titration of Luciferase-expressing Recombinant Viruses. J. Vis. Exp. (91), e51890, doi:10.3791/51890 (2014).

This video demonstrates the preparation of cell culture samples to assess viral load using a luciferase-based assay. It outlines the process of infecting epithelial cells with a genetically modified virus, transferring viral supernatants and standards into assay wells, and promoting viral entry through centrifugation and incubation. The expression of luciferase serves as a reporter, allowing quantification of viral concentration based on luminescent signal output.

Protocol

1. Sample Preparation

1. Obtain samples containing luciferase-expressing virus (herein VSV∆51-Fluc as an example) for titration and transfer into 96-well plates. Alternatively, perform infections in 96-well tissue culture plates and use supernatants directly.
2. Leave two columns untreated for the inclusion of standard curves. For experiments done directly in 96-well plates, titer at the end of the experiment (40 hr post-infection) or store at -80 °C and titer at a later date.

2. Preparation of Permissive Cells for Virus Titration

1. 24 hr prior to titering, prepare a suspension of Vero cells at a concentration of 2.5 x 10⁵ cells/ml in Dulbecco’s modified eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and 1% penicillin/streptomycin. 
   NOTE: Although this protocol uses Vero cells, any suitable permissive cell line for vesicular stomatitis virus (VSV) infection could be used.
2. Seed 2.5 x 10⁴ cells (100 μl) in 96-well white solid flat-bottom plates using a microplate dispenser.
   1. Prepare 12 ml of cell suspension per plate, plus 5 ml for priming.
   2. Clean the microplate dispenser cassette by flushing the tubing with 50 ml of sterile water.
   3. Fill the microplate dispenser lines with cell suspension and let 5 ml of cell suspension flow through.
   4. Select a program that will dispense 100 μl in each well of a white-walled 96-well plate. Dispense, and repeat as necessary for additional plates. Also, seed a few wells in a 96-well clear flat-bottom plate if opaque-bottom white-walled plates are used for verification of cell health and density.
   5. When finished, flush cells back into the original container. Clean the cassette by running 50 ml of 70% ethanol, followed by 50 ml of warm sterile water through the tubing.
   6. Make sure the cassette and tubing are appropriately cleaned between uses.
3. Incubate cells for 24 hr at 37 °C in a humidified 5% CO₂ incubator.

3. Preparation of Viral Standard Curve

1. Prepare a standard curve of VSV∆51-Fluc in serum-free DMEM such that the final concentration of plaque forming units (pfu) per ml after transfer onto Vero cells is as follows: 10⁸ pfu/ml, 10⁷ pfu/ml, 10⁶ pfu/ml, 10⁵ pfu/ml, 10⁴ pfu/ml, 10³ pfu/ml, 10² pfu/ml, and 10¹ pfu/ml. Prepare 50 μl of each concentration per plate of Vero cells plus an extra 10%.
   NOTE: Titer of the luciferase virus stock will need to be assessed in a classical way in order to generate a standard curve that will allow for absolute quantification. Otherwise, relative quantification can be achieved without precise titer information by arbitrarily setting the viral titer based on dilution steps. For example, the first dilution of the standard curve may be set to 10⁸ viral units, and the following 1/10 dilution to 10⁷, and so on. In this context, one should express values as a fold-change compared to a pre-determined sample, as absolute quantification will not be accurate.

4. Transfer of Sample Supernatants onto Permissive Cells

1. Check Vero cells plated in the clear-bottom 96-well plate under a light microscope to confirm that monolayers are at least 95% confluent.
2. Transfer 25 μl of sample supernatant onto Vero cells seeded in white-walled plates. Do not transfer supernatants into the 2 columns designated for standard curves. 
   NOTE: This can be done simultaneously for all wells on a single plate using a 96-channel liquid handler.
3. Using an 8- or 12-channel multi-channel pipettor, add 25 μl of each dilution of the standard curve prepared in Step 3 to the Vero cells in the 2 designated columns.
4. Centrifuge plates for 5 min at 430 x g at room temperature (RT).
5. Incubate for 5 hr at 37 °C in a humidified 5% CO₂ incubator.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbeccos' Modified Eagle's Medium (DMEM)	Corning	SH30243.01
Fetal bovine serum	NorthBio Inc.	NBSF-701
Phosphate buffered saline	Corning	21-040-CV
HEPES	Fisher Scientific	BP310-1	Prepare a 1 mM solution, pH 7.3
96-well solid white flat bottom polystyrene TC-treated microplates	Corning	3917	384-well plates can also be used for higher throughput
Synergy Mx	BioTek	SMTBL	Monochromator microplate reader
Liquidator96	Mettler Toledo	LIQ-96-200	96 tip manual pipetting system
Liquidator96 LTS Tips sterilized with filters	Mettler Toledo	LQR-200F	Any sterile filtered tips compatible with pipettors of choice are appropriate
Microflo	BioTek	111-206-21	Used to plate cells in a 96-well plate
Fluoroskan Ascent FL	Thermo Scientific	5210450	Microplate fluorometer