Determining Optimal Target Cell Density Using a Luciferase-Based Pseudovirus Infection Assay

Abstract

Source: Chang, X., et al. Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity. J. Vis. Exp. (2021)

This video demonstrates a luciferase-based pseudovirus titration assay to determine the optimal target cell density for maximum infection. By comparing luminescence across cell densities, the method ensures high assay sensitivity and reproducibility for downstream applications.

Protocol

1. Pseudovirus titration

1. Make single-cell suspensions of Madin-Darby canine kidney (MDCK) cells (5 x 10⁴ per mL) in complete Dulbecco's modified Eagle medium (DMEM medium), and add 1250, 2500, 5000, 10000, 20000, and 40000 cells to different wells of a 96-well flat-bottom plate. Incubate the cells in a 37 °C, 5% CO₂ incubator for 20 h.
2. Take out the pseudovirus from the -80 °C refrigerator, thaw it on the ice, and vortex the pseudovirus.
3. Add 6 HAU (6x Hemagglutinating units) pseudoviruses to each well of the 96-well round-bottom plate and replenish each well with complete DMEM up to 120 µL.
4. Pipett....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96-well cell culture plates (flat-bottom)	Thermo fisher scientific	167008	Consumable material
96-well cell culture plates (round-bottom)	Thermo fisher scientific	163320	Consumable material
Allegra X-15R	Beckman coulter	--	Equipment/Centrifuge
Dulbecco’s Modified Eagle Medium (DMEM)	Gibco	12100-046	Reagent
Luciferase Assay System Freezer Pack	Promega	E4530	Reagent kit
MDCK.1	ATCC	CRL-2935	Cell line
Microcentrifuge Tubes 1.5 mL	Thermo fisher scientific	509-GRD-Q	Consumable material
Pipette Tips (10 μL)	Thermo fisher scientific	TF102-10-Q	Consumable material
Pipette Tips (100 μL)	Thermo fisher scientific	TF113-100-Q	Consumable material
Pipette Tips (1000 μL)	Thermo fisher scientific	TF112-1000-Q	Consumable material