tetano
Editor, Senior Moderator
Methods Protoc
. 2021 Jan 7;4(1):E7.
doi: 10.3390/mps4010007.
Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures
Kaitlyn A Barrow[SUP] 1 [/SUP], Lucille M Rich[SUP] 1 [/SUP], Elizabeth R Vanderwall[SUP] 1 [/SUP], Stephen R Reeves[SUP] 1 2 [/SUP], Jennifer A Rathe[SUP] 3 [/SUP], Maria P White[SUP] 1 [/SUP], Jason S Debley[SUP] 1 2 [/SUP]
Affiliations
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[SUP]?[/SUP], 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.
Keywords: COVID-19; RNA; SARS-CoV-2; Vero E6; airway; epithelial; heat; inactivation; protein.
. 2021 Jan 7;4(1):E7.
doi: 10.3390/mps4010007.
Inactivation of Material from SARS-CoV-2-Infected Primary Airway Epithelial Cell Cultures
Kaitlyn A Barrow[SUP] 1 [/SUP], Lucille M Rich[SUP] 1 [/SUP], Elizabeth R Vanderwall[SUP] 1 [/SUP], Stephen R Reeves[SUP] 1 2 [/SUP], Jennifer A Rathe[SUP] 3 [/SUP], Maria P White[SUP] 1 [/SUP], Jason S Debley[SUP] 1 2 [/SUP]
Affiliations
- PMID: 33430421
- DOI: 10.3390/mps4010007
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[SUP]?[/SUP], 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.
Keywords: COVID-19; RNA; SARS-CoV-2; Vero E6; airway; epithelial; heat; inactivation; protein.