Human embryonic kidney (HEK) 293T CRL-3216 cells were purchased from American Type Culture Collection. All cells are regularly tested and are mycoplasma free. HEK293T and HeLa cell lines were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS), 2 mM l -glutamine, penicillin (100 U/ml), and streptomycin (100 mg/ml; Gibco) at 37°C with 5% CO2. The MT4 and SupT1 T cell lines were maintained in RPMI 1640 with l -glutamine (Corning) and supplemented with 10% FBS (GenClone), penicillin (100 U/ml), and streptomycin (100 mg/ml). Replication-deficient vesicular stomatitis virus glycoprotein (VSV-G)–pseudo-typed HIV-1 virions were produced in HEK293T cells using the packaging plasmid pMDG2, which encodes VSV-G envelope (Addgene plasmid no. 12259), pNL4-3–derived pCRV GagPol (HIV-1 clade B) (25 (link)), and pCSGW (26 (link)) as described previously (27 (link)). Mutagenesis of CA was performed using the QuickChange method (Stratagene) against pCRV GagPol, and primer sequences are given in table S1. The HIV-1 clade B infectious molecular clone pNL4-3 was used for all passage and virus release experiments. Mutant constructs were generated with the New England Biolabs (NEB) Q5 site directed mutagenesis kit (NEB, E0554), and primers (see table S1) were designed using the NEBaseChanger online tool.
Hek 293t crl 3216 cells
Manufactured by American Type Culture Collection
HEK) 293T CRL-3216 cells are a widely used human embryonic kidney cell line derived from human embryonic kidney cells. These cells are commonly used in research applications, such as the production of viral vectors, protein expression, and cell-based assays.
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Lab products found in correlation
Streptomycin, by Thermo Fisher Scientific (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Neb q5 site directed mutagenesis kit, by New England Biolabs (2 mentions)
Rpmi 1640 with l glutamine, by Corning (1 mentions)
Hcov 229e vr 740, by American Type Culture Collection (1 mentions)
Hcov oc43 vr 1558, by American Type Culture Collection (1 mentions)
Rd ccl 136, by American Type Culture Collection (1 mentions)
Pen strep, by Thermo Fisher Scientific (1 mentions)
Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Corning (1 mentions)
Rpmi 1640, by Corning (1 mentions)
3 protocols using hek 293t crl 3216 cells
1
Generating HIV-1 Virions in HEK293T Cells
2
SARS-CoV-2 Cell Culture and Reagents
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HuH-7 and Caco-2 cells were obtained from the Cell Bank of the Chinese Academy of Science. HEK-293T (CRL-3216™) cells and RD (CCL-136™) were obtained from the American Type Culture Collection (ATCC). All these cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin. HCoV-OC43 (VR-1558) and HCoV-229E (VR-740) were obtained from ATCC and amplified in HCT-8 and HuH-7 cells, respectively. His-tagged human ACE2, Fc-tagged RBD and Fc-tagged S1 subunit were obtained from Kactus Biosystems. EK1 peptide was synthesized by KareBay Biochem with a purity of > 95%.
3
HEK293T and THP-1 Cell Culture Protocols
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Human embryonic kidney (HEK) 293T CRL‐3216 cells were purchased from American Type Culture Collection. All cells are mycoplasma free and they are regularly tested. HEK293T was cultured in Dulbecco's modified Eagle's medium with 10% foetal bovine serum (FBS), 2 mM L‐glutamine, penicillin (100 U/ml) and streptomycin (100 mg/ml; Gibco) at 37°C with 5% CO2.
THP‐1 cells were maintained in RPMI (Gibco) supplemented with 10% FBS and Pen/Strep. THP‐1‐IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously (Mankan et al, 2014 (link)). THP‐1 Dual Control and STING KO and MAVS KO cells were previously described (Sumner et al, 2020 (link)). Jurkat and SupT1 T‐cell lines and PBMCs were maintained in RPMI 1640 with L‐glutamine (Corning) and supplemented with 10% FBS (GenClone), penicillin (100 U/ml) and streptomycin (100 mg/ml). STING inhibitor H151 was obtained from Invitrogen.
Mutant construct D185E was generated with the New England Biolabs (NEB) Q5 site‐directed mutagenesis kit (NEB, E0554) against pCRV GagPol WT and mutants (K158A, K158A/T8I, T8I; Mallery et al, 2021 ) using primers designed using the NEBaseChanger online tool.
THP‐1 cells were maintained in RPMI (Gibco) supplemented with 10% FBS and Pen/Strep. THP‐1‐IFIT1 cells that had been modified to express Gaussia luciferase under the control of the IFIT1 promoter were described previously (Mankan et al, 2014 (link)). THP‐1 Dual Control and STING KO and MAVS KO cells were previously described (Sumner et al, 2020 (link)). Jurkat and SupT1 T‐cell lines and PBMCs were maintained in RPMI 1640 with L‐glutamine (Corning) and supplemented with 10% FBS (GenClone), penicillin (100 U/ml) and streptomycin (100 mg/ml). STING inhibitor H151 was obtained from Invitrogen.
Mutant construct D185E was generated with the New England Biolabs (NEB) Q5 site‐directed mutagenesis kit (NEB, E0554) against pCRV GagPol WT and mutants (K158A, K158A/T8I, T8I; Mallery et al, 2021 ) using primers designed using the NEBaseChanger online tool.
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