HEK 293T cells (CRL-3216; ATCC, Manassas, VA, USA) and the mouse monocyte/macrophage cell line J774A.1 (TIB-67; ATCC, Manassas, VA, USA) were each cultured in Dulbecco’s Minimal Essential Media (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 10 mM HEPES, 100 U/mL penicillin and 100 μg/mL streptomycin (all from Corning, Tewksbury, MA, USA). The human monocyte cell line, THP-1 (TIB-202; ATCC, Manassas, VA, USA) was cultured in RPMI-1640 and the same supplements as well as 0.05 mM 2-mercaptoethanol (Sigma, St Louis, MO, USA). HEK-Blue-hTLR5 cells (Invivogen, San Diego, CA, USA) were cultured in Dulbecco’s Minimal Essential Media (DMEM) supplemented with 10% FBS, 10 mM HEPES, 50 U/mL penicillin, 50 μg/mL streptomycin, 30 μg/mL blasticidin and 100 μg/mL zeocin (the latter two reagents from Invivogen, San Diego, CA, USA). All cells were cultured at 37 °C and 5% CO2 in a humidified incubator.
Hek293t cells crl 3216
Manufactured by American Type Culture Collection
Sourced in United States
HEK293T cells (CRL-3216) are a commonly used human cell line derived from human embryonic kidney cells. They are transformed with the adenovirus E1A and E1B genes, which can induce cell immortalization and facilitate high levels of protein expression. These cells are suitable for a variety of applications, including gene expression, virus production, and drug screening.
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Lab products found in correlation
Formic acid, by Thermo Fisher Scientific (3 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Penicillin, by InvivoGen (1 mentions)
Streptomycin, by InvivoGen (1 mentions)
Hek blue htlr5 cells, by InvivoGen (1 mentions)
Rpmi 1640, by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Zeocin, by InvivoGen (1 mentions)
Blasticidin, by InvivoGen (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Polyethylenimine pei 25kda linear polymer, by Polysciences (1 mentions)
Hek293t cells, by Thermo Fisher Scientific (1 mentions)
A549 hace2tpsa, by InvivoGen (1 mentions)
Restriction endonuclease, by Takara Bio (1 mentions)
High fidelity dna polymerase, by Takara Bio (1 mentions)
Chromatographic grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
δ aminolevulinic acid, by Merck Group (1 mentions)
13 protocols using hek293t cells crl 3216
1
Cell Culture Protocols for Immune Research
2
JRFL Env Plasmid Transfection in HEK293T Cells
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The plasmid encoding WT JRFL Env was a kind gift from Dr. Joseph Sodroski. HEK293T cells (CRL-3216, human embryonic kidney 293 cells with SV40 T-antigen expression) were obtained from ATCC (Manassas, VA, USA). Polyethylenimine (PEI) 25kDa linear polymer was obtained from Polysciences, Inc. (Warrington, PA, USA). All other reagents, unless otherwise noted, were obtained from Fisher Scientific (Hampton, NH, USA).
HEK293T cells were seeded at 3 million cells per T75 flask, transiently transfected with 4 µg of JRFL Env plasmid DNA and 48 µL PEI (1 mg/mL solution), adapted from the pseudovirus production protocol used in prior studies [22 (link),23 (link),26 (link),28 (link)]. Cells were detached with 5 mM EDTA in DPBS at 24 h after transfection and reseeded at 50,000 cells per well in 48-well plates. Transfection efficiency was measured at 90–95% by flow cytometry.
HEK293T cells were seeded at 3 million cells per T75 flask, transiently transfected with 4 µg of JRFL Env plasmid DNA and 48 µL PEI (1 mg/mL solution), adapted from the pseudovirus production protocol used in prior studies [22 (link),23 (link),26 (link),28 (link)]. Cells were detached with 5 mM EDTA in DPBS at 24 h after transfection and reseeded at 50,000 cells per well in 48-well plates. Transfection efficiency was measured at 90–95% by flow cytometry.
3
Isolation and Culture of Lung Spheroid Cells
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Lung spheroid cells (LSC) were isolated from lung samples of healthy human obtained from the National Disease Research Interchange and passaged every 3–5 days, which has been conducted in our previous studies32 (link). After 2–3 passages, LSC were plated on a fibronectin-coated flask and maintained in Iscove’s Modified Dulbecco’s Media (IMDM) containing 20% fetal bovine serum (FBS). Media changes were performed every other day. LSC were allowed to reach 70–80% confluence before generating serum-free secretome (LSC-Secretome). LSC-Secretomes were collected and filtered with a 0.22 μm filter to remove cellular debris. HEK293T cells (CRL-3216) were purchased from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 10% FBS. A549 cells expressing human ACE2 and human TMPRSS2 were purchased from InvivoGen (a549-hace2tpsa) and cultured in DMEM with 10% FBS. All procedures in this study were in accordance with the ethical standards of the institutional research committee and with the guidelines set by the Declaration of Helsinki.
4
Recombinant Protein Expression Protocol
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AFB1 standard was purchased from Qingdao Pribolab Bioengineering Limited Corporation (Qingdao, China). Restriction endonucleases and high-fidelity DNA polymerase were purchased from TaKaRa Biomedical Technology Corporation (Beijing, China). A QuikChange site-directed mutagenesis kit was purchased from Stratagene (Guangzhou, China). δ-aminolevulinic acid was purchased from Sigma Aldrich (St. Louis, MO, USA). Isopropyl-β-D-thiogalactoside (IPTG), NADPH, and BCA protein quantitation kits were purchased from the Beijing Dingguo Changsheng Biotechnology Limited Corporation. A 5 mL HisTrap HP column was purchased from GE healthcare (Uppsala, Sweden). A ZORBAX SB-C18 column (3.0 × 100 mm, 1.8 microns) was purchased from Agilent (Santa Clara, CA, USA). HEK293T cells (CRL-3216) were purchased from ATCC. Chromatographic grade acetonitrile, methanol, and formic acid were purchased from Thermo Fisher Scientific (Rockford, IL, USA). All primers were synthesized in Invitrogen (Guangzhou, Guangdong province, China). All the other reagents and chemicals used were of the highest analytical grade available.
5
Luciferase Reporter Assay in HEK293T Cells
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HEK293T cell luciferase reporter assays were performed as previously described (17 (link)). Briefly, cells were transfected following the calcium phosphate transfection protocol in 6-well plates. Experimental plasmids and usage amounts are described in Supplementary Table 1 . 48 hours after transfection, cells were harvested, digested, and assayed for luciferase activity using the Dual-Luciferase Reporter Assay System (Promega) following the manufacturer’s protocol. Data were collected with a TD-20/20 Luminometer (Turner Designs) and processed with customized python scripts.
The HEK293T cells (CRL-3216™) were obtained directly from ATCC (American Type Culture Collection) and used within one year of purchase. The cells were tested negative for mycoplasma contamination.
The HEK293T cells (CRL-3216™) were obtained directly from ATCC (American Type Culture Collection) and used within one year of purchase. The cells were tested negative for mycoplasma contamination.
6
Cell Line Culturing Procedures
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Details on the cell and molecular experimental procedures used are described in the Supplementary Materials and Methods . Human hepatocellular carcinoma (HepG2) cells (HB-8065) and human embryonic kidney (HEK) 293 T cells (CRL-3216) were obtained from the American Type Culture Collection (ATCC, USA). SNU387 cells (CRL-2237) were obtained from the Korean Cell Line Bank (KCLB, Republic of Korea).
7
Cultivation of Porcine Cell Lines for ASFV Studies
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The HEK-293T cells (CRL-3216) and PK-15 cells (CCL-33) were acquired from the American Type Culture Collection. The generous provision of wild boar lung (WSL) cells was made by Professor Guiqing Peng of Huazhong Agricultural University in Wuhan, China. The cells were cultivated under carefully controlled conditions at a temperature of 37°C with a 5% CO2 atmosphere. They were nourished in Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS). The primary porcine alveolar macrophage (PAM) cells were prepared from bronchoalveolar lavage according to previously described methods (41 (link)) and cultured in RPMI-1640 medium supplemented with 10% FBS. The SP2/0 cells were cultured in RPMI-1640 medium with 10% FBS. The genotype II ASFV isolate strain CN/SD/2019 was propagated in PAM cells.
8
Characterization of Colorectal Cancer Cell Lines
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HT-29 cells (HTB-38), Caco-2 cells (HTB-37), and HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection (Manassas, VA, USA). HCT116 mlh1-2 cells, stably transfected with the pcDNA3.1+/MLH1 overexpression plasmid, were a gift from Professor Franҫoise Praz (Centre National de la Recherche Scientifique in Paris, France). Cells were grown in DMEM (Thermo Fisher Scientific; Waltham, MA, USA) with 10% FBS (Sigma-Aldrich; St. Louis, MO, USA) and 1% penicillin-streptomycin (Sigma-Aldrich). The growth media of stably transduced cell lines was supplemented with puromycin; 5 μg/mL for HT-29 and Caco-2 cells, and 4 μg/mL for HCT116 mlh1-2 cells. All cell lines were regularly tested for mycoplasma, and STR profiling was carried out as recently described [21 (link)]. The CRC cell lines used in this study carry different combinations of recurrent genetic and epigenetic alterations: HT-29 cells are TP53-mutant, KRAS wild-type, BRAF-mutant, MSS, CIMP-positive; Caco-2 cells are TP53-mutant, KRAS wild-type, BRAF wild-type, MSS, CIMP-negative; HCT116 mlh1-2 cells are TP53 wild-type, KRAS-mutant, BRAF wild-type, MSI, CIMP-positive [22 (link)].
9
Mammalian Cell Culture and Plasmid Expression
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Human embryonic kidney HEK293T cells (CRL-3216) and human colorectal adenocarcinoma SW480 (CCL-228) and HCT116 cells (CCL-247) were obtained from American Type Culture Collection. HEK293T cells were maintained in DMEM (Corning) supplemented with 10% FBS (Gibco) at 37°C in a humidified, 5% CO2 incubator. HCT116 cells were maintained in McCoy’s 5a Modified Medium (Corning) supplemented with 10% FBS, while SW480 cells were maintained in Leibovitz’s L-15 Medium (Corning) supplemented with 10% FBS without CO2. Cell transfection was conducted with VigoFect (Vigorous Biotechnology) or Lipofectamine 2000 (Invitrogen).
All plasmids used in this study were generated by subcloning corresponding cDNAs into HA-pcDNA3.1 (for mammalian cell expression), pET32M.3C, pETMBP.3C (for bacteria expression), or pCS107-HA (for in vitro transcription) vectors. The sequences encoding human Axin1 (NCBI RefSeq no. NM_181050.3), human APC (NM_000038.6), human GSK3β (NM_001146156.2), β-catenin (NM_001098209.2), and human CK1α (NM_001025105.3) were amplified using standard PCR procedures with cDNA from HEK293T cells and subcloned into HA-pcDNA3.1, pETMBP.3C, and pCS107-HA vectors. Drosophila melanogaster dAPC2 (NM_001347814.1) was amplified and subcloned into pETMBP.3C vectors. Plasmids encoding Axin1 deletions or mutations were generated by PCR using primers with appropriate nucleotide substitutions.
All plasmids used in this study were generated by subcloning corresponding cDNAs into HA-pcDNA3.1 (for mammalian cell expression), pET32M.3C, pETMBP.3C (for bacteria expression), or pCS107-HA (for in vitro transcription) vectors. The sequences encoding human Axin1 (NCBI RefSeq no. NM_181050.3), human APC (NM_000038.6), human GSK3β (NM_001146156.2), β-catenin (NM_001098209.2), and human CK1α (NM_001025105.3) were amplified using standard PCR procedures with cDNA from HEK293T cells and subcloned into HA-pcDNA3.1, pETMBP.3C, and pCS107-HA vectors. Drosophila melanogaster dAPC2 (NM_001347814.1) was amplified and subcloned into pETMBP.3C vectors. Plasmids encoding Axin1 deletions or mutations were generated by PCR using primers with appropriate nucleotide substitutions.
10
Culture and Maintenance of HEK293T, hVSMCs, and HTLA Cells
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