Home › Microbiology › High-throughput Titration of Luciferase-expressing Recombinant Viruses
Microbiology JoVE (Open Access) Citable · DOI

High-throughput Titration of Luciferase-expressing Recombinant Viruses

DOI: 10.3791/51890-v
What you'll learn
  • ✓ Perform high-throughput viral titration using luciferase expression assays
  • ✓ Prepare permissive cells and viral standard curves for automated quantification
  • ✓ Analyze bioluminescence data to determine viral titers and cytotoxicity
Protocol

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Difficulty
advanced
Total time
~4–6 hours per experiment (including cell preparation, viral incubation, and luminescence readout)
Model organism
HEK293 or permissive mammalian cell lines
Biosafety
BSL-2

Steps

1
Prepare permissive cells for virus titration

Culture and prepare permissive cells (e.g., HEK293) in appropriate growth medium. Ensure cells are viable and at suitable confluence for viral infection assays.

▶ 01:16
2
Prepare viral standard curve and transfer samples

Generate serial dilutions of luciferase-expressing viral standard and transfer sample supernatants onto permissive cell cultures using automated or manual liquid handlers.

▶ 02:57
3
Assess cell viability and prepare luciferin substrate

Monitor cell viability following viral exposure using appropriate assays. Prepare and equilibrate luciferin substrate reagent for bioluminescence measurement.

▶ 04:08
4
Read bioluminescence and analyze viral titer data

Perform bioluminescence measurements using a luminometer. Process raw luminescence values against the standard curve to quantify viral titers and assess sample cytotoxicity.

▶ 04:53
💬 Comments coming soon