Overview
In this video, we demonstrate the cytopathic effect assay to determine the tissue culture infective viral dose of Drosophila C virus on Drosophila S2* cells. A range of viral concentrations are used to infect the cultured cells. The proportion of cells exhibiting structural changes is determined, and this data is used to calculate the tissue culture infective dose.
Protocol
1. Drosophila C virus load measure by CPE assay
- Pick 5 random virus-containing tubes to determine the average virus tissue culture infective dose (TCID50) by a cytopathic effect (CPE) assay (1 unit of TCID50= 0.7 plaque-forming unit [PFU]).
NOTE: CPE refers to structural changes in host cells caused by viral invasion. The infecting virus causes lysis of the host cell or when the cell dies without lysis due to an inability to reproduce. - On day 1, collect prepared S2* cells by pipetting. Count the cell number. Centrifuge cells for 300 x g for 4 min and discard the supernatant. Dilute the cells to the density of 1 x 106 cells/mL with a complete medium for S2* cells.
- Seed 1 x 105 S2*cells (100 μL) per well to the flat-bottom 96-well-plate (~30% confluency).
- Perform serial 10-fold dilutions of the DCV stock with complete medium for S2* cells. Add 50 µL dilutions into the well. Add 50 µL of culture medium without virus to the well classified as the negative control well.
NOTE: For each dilution rate, 8 wells were used. As the typical DCV yield is around 108-109 PFU/mL, the dilution should at least cover ~10−5-10−10. - On day 4, observe CPE with a bright-field microscope with a 20X or 40X objective. Classify a well in which the cells look blurry and the medium is full of fragments as a "positive well", and a well in which the cell morphology is normal as a "negative well".
- Mark CPE positive or negative wells with + or –, respectively. Calculate the virus TCID50 by the Reed and Muench method.
Subscription Required. Please recommend JoVE to your librarian.
Materials
| Name | Company | Catalog Number | Comments |
| 1.5 ml Microcentrifuge tubes | Brand | 352070 | |
| 10 cm cell culture dish | Sigma | CLS430167 | Cell culture |
| Cell Incubator | Sanyo | MIR-553 | |
| Centrifuge | Eppendorf | 5810R | |
| Centrifuge | Eppendorf | 5424R | |
| Drosophila Incubator | Percival | I-41NL | Rearing Drosophila |
| FBS | Invitrogen | 12657-029 | Cell culture |
| Schneider's Insect Medium | Sigma | S9895 | Cell culture |
| Statistical software | GraphPad Prism 7 | ||
| Flat bottom 96-well-plate | Sigma | CLS3922 | Cell culture |
| Cell Incubator | Sanyo | MIR-553 | |
| Microscope | Olympus | CKX41 |
