A Focus-Forming Assay for Quantifying Virus Titers in the Organs of an Infected Mouse
Published: January 31, 2024
Abstract
Source: Brien, J. D. et al., Isolation and Quantification of Zika Virus from Multiple Organs in a Mouse. J. Vis. Exp. (2019)
The video demonstrates a procedure for quantifying the Zika virus in multiple mouse organs via a focus-forming assay. The process involves infecting mammalian cell monolayers with virus-infected tissue homogenates, followed by the use of methyl cellulose to prevent viral spread. Subsequently, immunostaining is employed to enable precise quantification and measurement of organ-specific infectivity.
Protocol
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Organ Isolation NOTE: The virus is not stable at room temperature (RT) so the number of animals harvested at one time must be planned carefully to preserve viral titers. Infect mice using the chosen dose and route, based on the phenotype required. For this protoc…
Declarações
The authors have nothing to disclose.
Materials
| 1-bromo-3-chloropropane (BCP) | MRC gene | BP151 | |
| 10cc syringe | Thermo Fisher Scientific | BD 309642 | |
| 18G needle | Thermo Fisher Scientific | 22-557-145 | |
| 1cc TB syringe | Thermo Fisher Scientific | 14-823-16H | |
| 20cc syringe | Thermo Fisher Scientific | 05-561-66 | |
| 24 tube beadmill | Thermo Fisher Scientific | 15 340 163 | |
| 3.2 mm stainless steel beads | Thermo Fisher Scientific | NC9084634 | |
| 37C Tissue Culture incubator | Nuair | 5800 | |
| 4G2 antibody | in house | ||
| 96 well flat bottom plates | Midsci | TP92696 | |
| 96well round bottom plates | Midsci | TP92697 | |
| Basix 1.5ml eppendorf tubes | Thermo Fisher Scientific | 02-682-002 | |
| Concentrated Germicidal Bleach | Staples | 30966CT | |
| CTL S6 Analyzer | CTL | CTL S6 Universal Analyzer | |
| Curved cutting scissors | Fine Science Tools | 14061-11 | |
| Dulbecco's Modified Eagle's Medium – high glucose With 4500 mg/L glucose | MilliporeSigma | D5671 | |
| Ethanol (molecular biology-grade) | MilliporeSigma | e7023 | |
| Fetal Bovine Serum | MilliporeSigma | F0926-500ML | |
| Forceps | Fine Science Tools | 11036-20 | |
| Glacial acetic acid | MilliporeSigma | 537020 | |
| Goat anti-mouse HRP-labeled antibody | MilliporeSigma | 8924 | |
| HEPES 1 M | MilliporeSigma | H3537-100ML | |
| Isopropanol (molecular biology-grade) | MilliporeSigma | I9516 | |
| Ketamine/Xylazine cocktail | Comparative Medicine | ||
| L-glutamine | MilliporeSigma | g7513 | |
| Magmax RNA purification kit | Thermo Fisher Scientific | AM1830 | |
| Methylcellulose | MilliporeSigma | M0512 | |
| Microcentrifuge | Ependorf | 5424R | |
| MiniCollect 0.5ml EDTA tubes | Bio-one | 450480 | |
| O-ring tubes | Thermo Fisher Scientific | 21-403-195 | |
| One step q RT-PCR mix | Thermo Fisher Scientific | 4392938 | |
| Paraformaldehyde | Thermo Fisher Scientific | EMS- 15713-S | |
| Phosphate Buffered Saline | MilliporeSigma | d8537-500ml | |
| Proline multichannel pipettes | Sartorius | 72230/72240 | |
| Proline single channel pipettes | Sartorius | 728230 | |
| RNAse free water | Thermo Fisher Scientific | 10-977-023 | |
| RNAzol BD | MRC gene | RB192 | |
| Rocking Platform | Thermo Fisher Scientific | 11-676-333 | |
| RPMI 1640 | Fisher | MT10040CV | |
| Saponin | MilliporeSigma | s7900 | |
| Spoon/Spatula | Fine Science Tools | 10090-17 | |
| Straight cutting scissors | Fine Science Tools | 14060-11 | |
| Triton X-100 | MilliporeSigma | t8787 | |
| True Blue Substrate | VWR | 95059-168 | |
| Trypsin | MilliporeSigma | T3924-100ML |
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Citar este artigo
A Focus-Forming Assay for Quantifying Virus Titers in the Organs of an Infected Mouse. J. Vis. Exp. (Pending Publication), e21929, doi: (2024).