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

Zusammenfassung

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.

Protokoll

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. Pipette up and down the mixture 5 times to mix thoroughly. Incubate the mixture plate in a 37 °C, 5 % CO₂ incubator for 1 hour.
5. Transfer 100 µL of the mixture to the cells in the 96-well plate. Put the cell culture plate back into the incubator for 48 h, 60 h, and 72 h after virus infection.
6. Take the plate out of the incubator and perform the luciferase assay.
7. Remove the medium from the wells of the plate carefully. Rinse the cells with 200 µL of phosphate-buffered saline (PBS) and remove PBS as much as possible.
   NOTE: Flip the plate and discard the supernatant by reverse-slapping it onto absorbent paper. If the testing cell line could not firmly attach to the bottom, remove the medium by aspiration carefully.
8. Add 50 µL of the lysis buffer to each well, and rock the culture plate several times. Store the plate at -80 °C overnight.
   NOTE: Mix 1 volume of 5x lysis buffer with 4 volumes of water to make the 1x lysis buffer. Equilibrate the 1x lysis buffer to room temperature (RT) before use. Rock the plate to ensure that the cells are covered with the lysis buffer completely. The single freeze-thaw process can help the cell be lysed completely.
9. Take the plate out, equilibrate at RT for 2 h.
   NOTE: The cell should be lysed completely.
10. Rock the plate several times gently, and transfer all the cell lysates to opaque 96-well plates.
11. Add 50 µL of the substrate to each well, rock the plate several times gently, and mix the substrate and lysis buffer thoroughly.
12. Measure the light produced within 5 min using a luminometer. Record the luciferase titers of the pseudovirus.
    NOTE: When a proper substrate is added, light is emitted as a by-product via a chemical reaction in which luciferin is converted to oxyluciferin by the luciferase enzyme. The amount of light produced is proportional to the amount of the luciferase enzyme.

Materialien

Liste der in diesem Artikel verwendeten Materialien

Name	Unternehmen	Katalognummer	Kommentare
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