SARS-CoV-2 Infection Assay in HEK293-ACE2 Cells

The following reagents were purchased from the indicated manufacturers: Lipofectamine 2000 (Invitrogen); puromycin (Thermo); Dual-specific luciferase assay kit (Promega); SYBR (BIO-RAD); polybrene (Millipore); MTT (MedChemExpress); ELISA kit for human IFN-β and TNF-α (4A Biotech); mouse antibodies against Flag and β-actin (Sigma), His and HA (OriGene), MyC (9B11), and IKKβ (8943S) (Cell Signaling Technology); rabbit monoclonal antibodies against HA, phosphor-IKKα (Ser176)/β (Ser177) (2078S) (Cell Signaling Technology), phosphor TBK1 (ab109272) and TBK1 (ab40676) (Abcam); and rabbit polyclonal antibodies against MAVS (Bethyl). HEK293, HeLa and HEK293T cells were purchased from ATCC. HEK293 cells stably expressing ACE2 (HEK293-ACE2) were obtained by lentiviral transduction. SeV (Cantell strain) was obtained as previously described.^{40 (link)} SARS-CoV-2 (IVCAS 6.7512) was isolated from BALF collected from a patient with viral pneumonia in December 2019 in Wuhan, China.^{41 (link)} Isolated viral particles were propagated in Vero E6 cells.^{42 (link)} HEK293-ACE2 cells were infected with SARS-CoV-2 in a biosafety level 4 (BSL-4) laboratory (Wuhan).

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Fu Y.Z., Wang S.Y., Zheng Z.Q., Yi H.u.a.n.g., Li W.W., Xu Z.S, & Wang Y.Y. (2020). SARS-CoV-2 membrane glycoprotein M antagonizes the MAVS-mediated innate antiviral response. Cellular and Molecular Immunology, 18(3), 613-620.

Publication 2020

Lipofectamine 2000 puromycin Dual-specific luciferase assay kit polybrene MTT mouse antibodies against Flag and β-actin (ab109272)

Ace2 Actin Antibodies Assay Cells Elisa Hek293 cells Hela Human Ikkα Ikkβ Lipofectamine 2000 Luciferase Mavs Monoclonal antibodies Mouse Patient Phosphor Polybrene Promega Puromycin Rabbit Sars cov 2 Strain Tbk1 Tnf α Vero cells Viral particles Viral pneumonia

Corresponding Organization : Chinese Academy of Sciences

Other organizations : University of Chinese Academy of Sciences

Top 5 similar protocols

Variable analysis

independent variables

SARS-CoV-2 (IVCAS 6.7512) virus
HEK293-ACE2 cells

dependent variables

Viral propagation in Vero E6 cells

control variables

Lipofectamine 2000 (Invitrogen)
Puromycin (Thermo)
Dual-specific luciferase assay kit (Promega)
SYBR (BIO-RAD)
Polybrene (Millipore)
MTT (MedChemExpress)
ELISA kit for human IFN-β and TNF-α (4A Biotech)
Mouse antibodies against Flag, β-actin, His, HA, MyC, and IKKβ
Rabbit monoclonal antibodies against HA, phosphor-IKKα (Ser176)/β (Ser177), phosphor TBK1, and TBK1
Rabbit polyclonal antibodies against MAVS
HEK293, HeLa and HEK293T cells (ATCC)
SeV (Cantell strain)

positive control

HEK293-ACE2 cells infected with SARS-CoV-2 in a biosafety level 4 (BSL-4) laboratory (Wuhan)

negative control

Not explicitly mentioned

Annotations

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