As described before (27 (link)), Vero cells were infected with reovirus serotype 3 (VR-824, ATCC) at a multiplicity of infection (MOI) of 0.1 PFU/cell. The supernatant was collected 48 hrs post viral infection, centrifuged at 2500 rpm for 30 min, and then passed through 0.45 µm filters. The filtered supernatant was centrifuged at 21,000 rpm for 180 min. The viral pellet was resuspended in RLT Buffer (RNeasy kit, Qiagen), which was followed by RNA extraction according to the manufacturer’s manual.
Vr 824
Manufactured by American Type Culture Collection
The VR-824 is a compact and versatile laboratory equipment designed for various applications. It provides a stable and controlled environment for incubating and cultivating microbial cultures. The device features precise temperature regulation, ensuring consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Low attachment 6 well plate, by Corning (2 mentions)
Rneasy kit, by Qiagen (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Dld 1, by National Collection of Authenticated Cell Cultures (1 mentions)
Nci h460, by National Collection of Authenticated Cell Cultures (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
7 protocols using vr 824
1
Reovirus Infection and RNA Extraction
2
Reovirus Infection and Organ Analysis in Mice
3
Reovirus Propagation and Inactivation
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The murine fibroblastic cell line L929 was obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified eagle’s medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% (v/v) glutamine (Gibco) and 1% (v/v) penicillin/streptomycin (Gibco). The prostate cancer cell line PC-3, colorectal carcinoma cell line DLD-1, and large cell lung carcinoma cell line NCI-H460 were obtained from China Center for Type Culture Collection (CCTCC), and cultured in RPMI-1640 at 37°C, 5% CO2 with 95% humidity.
Reovirus type 3 Dearing strain was obtained from ATCC (VR-824) and stored in -80°C until use. Reovirus was propagated in L929 cells, titrated by a standard plaque assay. For generation of UV-inactivated reovirus, reovirus in PBS were exposed to UV light (shortwave 254nm) for 30 minutes. The UV-induced loss of reoviral replicability was confirmed with L929 cell viability assay.
Reovirus type 3 Dearing strain was obtained from ATCC (VR-824) and stored in -80°C until use. Reovirus was propagated in L929 cells, titrated by a standard plaque assay. For generation of UV-inactivated reovirus, reovirus in PBS were exposed to UV light (shortwave 254nm) for 30 minutes. The UV-induced loss of reoviral replicability was confirmed with L929 cell viability assay.
4
Reovirus Isolation and Characterization
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The wild-type reovirus strain R124 (here referred to as Reo) was previously isolated from a heterogeneous reovirus T3D stock (VR-824) obtained from the American Type Culture Collection by two rounds of plaque purification using HER911 cells.12 (link) Reovirus mutant Jin-3 was isolated from JAM-A-deficient U118MG cells after passaging of the wild-type T3D strain R124.12 (link) All experiments were performed using cesium chloride-purified stocks as described earlier.11 (link) The total amount of particles was calculated based on OD260 values where 1 OD260 equals 2.10×1012 reovirus particles/mL,13 (link) and the infectious titer was quantified by plaque assay on HER911 cells.14 (link)
5
Reovirus Purification and Inactivation
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The wild-type reovirus strain R124 (here referred to as reovirus) was previously isolated from a heterogeneous reovirus T3D stock (VR-824) obtained from the American Type Culture Collection (ATCC) by two rounds of plaque purification using HER911 cells.20 (link) All experiments were performed using cesium chloride-purified stocks (see online supplemental materials ). The total amount of particles was calculated based on optical density (OD)260 values, where 1 OD equals 2.10*1012 reovirus particles/mL.21 (link) The infectious titer was quantified by plaque assay on HER911 cells.22 (link) Reovirus particles were inactivated by exposure to shortwave ultraviolet light (254 nm) for 15 min at room temperature on a low-attachment 6-well plate (Corning).23 (link) Afterward, the total amount of viral particles was determined based on the OD260 values. A correction value was calculated to ensure an equal number of viral particles for treatments with infectious and UV-inactivated reovirus (UVi).
6
Isolation and Characterization of Reovirus R124
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The wild-type reovirus strain R124 (here referred to as reovirus) was previously isolated from a heterogeneous reovirus T3D stock (VR-824) obtained from the American Type Culture Collection (ATCC) by two rounds of plaque purification using HER911 cells. 20 (link) All experiments were performed using cesium chloride-purified stocks (see online supplemental materials). The total amount of particles was calculated based on optical density (OD)260 values, where 1 OD equals 2.10*10 12 reovirus particles/ mL. 21 (link) The infectious titer was quantified by plaque assay on HER911 cells. 22 (link) Reovirus particles were inactivated by exposure to shortwave ultraviolet light (254 nm) for 15 min at room temperature on a low-attachment 6-well plate (Corning). 23 (link) Afterward, the total amount of viral particles was determined based on the OD260 values. A correction value was calculated to ensure an equal number of viral particles for treatments with infectious and UV-inactivated reovirus (UVi).
7
Reovirus and PRV Infection in Fetal Rhesus Monkey Kidney Cells
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Fetal rhesus monkey kidney (TF-104) cells were grown in Eagle's minimum essential medium (EMEM) supplemented with 5% fetal bovine serum (FBS) and 100 U/mL penicillin, and 100 μg/mL streptomycin and 100 U/mL amphotericin B. Cells were maintained at 37 °C with 5% CO2. Antibiotic, trypsin-EDTA, FBS, and EMEM were supplied by Gibco BRL (Grand Island, NY, USA). The reovirus Type 1 (Lang, ATCC VR-230), Type 2 (Jones, ATCC VR-231), Type 3 (Dearing, ATCC VR-824) purchased from American Type Culture Collection (ATCC) and PRV (KRP113 strain) isolated from fecal samples of Korean diarrheic piglets were used in this study. Reoviruses were preactivated with 10 μg/mL trypsin (GIBCO Invitrogen Corporation, CA, USA) for 30 min at 37 °C before being inoculated onto confluent TF-104 cells and infected cells were maintained in the presence of 1 μg/mL trypsin (GIBCO Invitrogen Corporation, CA, USA).
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