Viral isolation was attempted for all RT-qPCR positive cases with available residual respiratory specimens, as described previously.49 (link) Briefly, VeroE6/TMPRSS2 cells50 (link) (JCRB1819, JCRB Cell Bank) were seeded in 96-well flat-bottom plates, respiratory specimens mixed with DMEM supplemented with 2% FBS and Antibiotic-Antimycotic Solution (Thermo Fisher Scientific) were inoculated in duplicate, and the culture supernatant was changed to fresh medium one day post-infection (d.p.i.) and was incubated at 37°C and supplied with 5% CO2. On one and five d.p.i., a cytopathic effect was observed. After five days, the supernatant was collected and RT-qPCR using the SARS-CoV-2 direct detection RT-qPCR kit (Takara) was performed in order to confirm the propagation of SARS-CoV-2. The median tissue culture infectious dose (TCID50) in the residual specimens was determined for all viral isolation positive cases.
Jcrb1819
Manufactured by Japanese Collection of Research Bioresources Cell Bank
JCRB1819 is a cell culture incubator that provides a controlled environment for cultivating cells. It maintains constant temperature, humidity, and carbon dioxide levels to support cell growth and proliferation. The core function of JCRB1819 is to provide an optimal and sterile environment for cell culture applications.
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Lab products found in correlation
Sars cov 2 direct detection rt qpcr kit, by Takara Bio (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions)
Geneticin selective antibiotic g418 sulfate, by Thermo Fisher Scientific (1 mentions)
2 protocols using jcrb1819
1
SARS-CoV-2 Viral Isolation and Quantification
2
Characterizing SARS-CoV-2 Variants in VeroE6-TMPRSS2 Cells
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The SARS-CoV-2 USA_WA1/2020 (SARS-CoV-2/human/USA/WA-CDC-02982586-001/2020, GenBank: MN985325.1) and B.1.1.529 (Omicron) sublineage BA.5.5 (hCoV-19/USA/COR-22-063113/2022, GISAID accession ID: EPI_ISL_13512579) were obtained from the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA, University of Texas Medical Branch, Galveston, TX, USA). Both viruses were passaged once in VeroE6-TMPRSS2 cells for subsequent experiments and deep sequenced to verify single nucleotide variants (SNVs) frequencies along the whole genome at the UTMB’s Next Generation Sequencing (NGS) Core, directed by Dr. Steven G. Widen. Deep sequencing of these stocks revealed no mutations or deletions in the Spike protein >5.0% frequency.
VeroE6 cell line expressing human TMPRSS2 were obtained from JCRB Cell Bank (JCRB1819, lot 04172020)36 (link). VeroE6-TMPRSS2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 100 µg/ml of Geneticin™ Selective Antibiotic - G418 Sulfate (Thermo Fisher Scientific). VeroE6-TMPRSS2 cell line was verified and tested negative for mycoplasma before use.
VeroE6 cell line expressing human TMPRSS2 were obtained from JCRB Cell Bank (JCRB1819, lot 04172020)36 (link). VeroE6-TMPRSS2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco/Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum and 100 µg/ml of Geneticin™ Selective Antibiotic - G418 Sulfate (Thermo Fisher Scientific). VeroE6-TMPRSS2 cell line was verified and tested negative for mycoplasma before use.
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