This video demonstrates the production of influenza pseudoviruses in human cells using calcium phosphate-mediated plasmid transfection, enabling safe generation of single-cycle viral particles for downstream applications such as antibody neutralization assays under biosafety level 2 conditions.
Method Article
Production of Pseudovirus Using Plasmid Transfection in Human Cells
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August 31st, 2026
In This Article
Abstract
Protocol
1. Pseudovirus packaging with calcium-phosphate transfection
- Make single-cell suspensions of HEK293FT cells (9 x 105 per mL) in complete DMEM (Dulbecco's Modified Eagle Medium) medium (Table 1). Add 10 mL of the single-cell suspension to a T75 flask, and incubate the cells in a 37 °C, 5% carbon dioxide (CO2) incubator for 20 h before the transfection.
NOTE: HEK293FT low passage (<20 passages) is recommended. Ensure that the cell monolayer is 80-90% confluent. - Replace the medium with 10 mL of fresh complete medium containing chloroquine (100 µM) 2 h before transfection.
- Mix the reagents and plasmid DNA as shown in Table 2 and Figure 1: ddH2O (double-distilled water), pCMV/R-HA, pCMV/R-NA, pHR-Luc, pCMV Δ8.9, 2.5 M CaCl2 (calcium chloride), and 2x 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer. Pipette up and down 5 times gently; incubate the mixture at room temperature (RT) for 20 min.
- Transfer the mixture into the medium above the cells, and rock the flask gently. Incubate the cells in the cell incubator for 15 h.
- Replace the medium with 15 mL of fresh complete DMEM medium. Incubate the cells in a 37 °C, 5% CO2 incubator for 65 h.
NOTE: The color of the medium will be light orange or slightly yellow, and at least 80% of the cell should show the cytopathic effect (CPE) under the inverted light microscope. - Harvest the supernatant, and centrifuge it at 2095 x g for 20 min at 4 °C. Collect the supernatant, aliquot it, and store it at -80 °C.
Table 1: Complete DMEM medium composition.
| Complete DMEM Medium Composition | Concentration |
| Dulbecco’s Modified Eagle Medium (DMEM) | 1x |
| Fetal bovine serum | 10% |
| Penicillin(1 U/mL)/Streptomycin | 1 μg/ mL |
Table 2: Pseudovirus packaging system.
| Materials | Concentration | Volume |
| 2x HEPES (pH 7.1) | -- | 675.0 μL |
| CaCl2 (2.5 M) | -- | 67.5 μL |
| ddH2O | -- | 529.2 μL |
| pCMV Δ 8.9 | 1000 ng/μL | 18.9 μL |
| pCMV/R-HA | 100 ng/μL | 27.0 μL |
| pCMV/R-NA | 50 ng/μL | 13.5 μL |
| pHR-Luc | 1000 ng/μL | 18.9 μL |
Results
Figure 1: Flowchart of Calcium-mediated transfection. This flowchart is used to describe the major procedures of calcium-mediated transfection.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Calcium chloride anhydrous | AMRESCO | 1B1110-500G | Reagent |
| Chloroquine diphosphate | Selleck | S4157 | Reagent |
| Dulbecco’s Modified Eagle Medium (DMEM) | Gibco | 12100-046 | Reagent |
| Fetal bovine serum | Gibco | 16000-044 | Reagent |
| HEK293FT | Gibco | R700-07 | Cell line |
| HEPES FREE ACID | AMRESCO | 0511-250G | Reagent |
| HIV-1 p24 antigen ELISA | ZeptoMetrix | 801111 | Reagent kit |
| Luciferase assay system freezer pack | Promega | E4530 | Reagent kit |
| Microcentrifuge tubes 1.5 mL | Thermo fisher scientific | 509-GRD-Q | consumable material |
| Nunc conical centrifuge tubes 15 mL | Thermo fisher scientific | 339650 | consumable material |
| Penicillin-streptomycin | Gibco | 15140-122 | Reagent |
| 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 |
| Serological pipettes (5 mL) | Thermo fisher scientific | 170355N | consumable material |
| Serological pipettes (10 mL) | Thermo fisher scientific | 170356N | consumable material |
