Back to Search
Start Over
Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures
- Source :
- Methods and Protocols, Methods and Protocols, Vol 4, Iss 7, p 7 (2021)
- Publication Year :
- 2020
-
Abstract
- Given that the airway epithelium is the initial site of infection, study of primary human airway epithelial cells (AEC) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will be crucial to improved understanding of viral entry factors and innate immune responses to the virus. Centers for Disease Control and Prevention (CDC) guidance recommends work with live SARS-CoV-2 in cell culture be conducted in a Biosafety Level 3 (BSL-3) laboratory. To facilitate downstream assays of materials from experiments there is a need for validated protocols for SARS-CoV-2 inactivation to facilitate safe transfer of material out of a BSL-3 laboratory. We propagated stocks of SARS-CoV-2, then evaluated the effectiveness of heat (65 °C) or ultraviolet (UV) light inactivation. We infected differentiated human primary AECs with SARS-CoV-2, then tested protocols designed to inactivate SARS-CoV-2 in supernatant, protein isolate, RNA, and cells fixed for immunohistochemistry by exposing Vero E6 cells to materials isolated/treated using these protocols. Heating to 65 °C for 10 min or exposing to UV light fully inactivated SARS-CoV-2. Furthermore, we found in SARS-CoV-2-infected primary AEC cultures that treatment of supernatant with UV light, isolation of RNA with Trizol®, isolation of protein using a protocol including sodium dodecyl sulfate (SDS) 0.1% and Triton X100 1%, and fixation of AECs using 10% formalin and Triton X100 1%, each fully inactivated SARS-CoV-2.
- Subjects :
- 0301 basic medicine
Vero E6
Letter
QH301-705.5
viruses
epithelial
Biochemistry, Genetics and Molecular Biology (miscellaneous)
Virus
Microbiology
03 medical and health sciences
chemistry.chemical_compound
0302 clinical medicine
Structural Biology
Viral entry
medicine
030212 general & internal medicine
inactivation
Sodium dodecyl sulfate
Biology (General)
skin and connective tissue diseases
Innate immune system
SARS-CoV-2
fungi
RNA
COVID-19
respiratory system
Epithelium
respiratory tract diseases
030104 developmental biology
medicine.anatomical_structure
chemistry
airway
Vero cell
Respiratory epithelium
heat
protein
Biotechnology
Subjects
Details
- ISSN :
- 24099279
- Volume :
- 4
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- Methods and protocols
- Accession number :
- edsair.doi.dedup.....0b60a1023c133bc57d66142b261f1db8