Anti–HLA-DP (HL-38, Sigma-Aldrich), anti–HLA-DP-DQ-DR (WR18, Bio-Rad), anti-rat RT1B (OX-6, BD Biosciences), anti–MHC class II (M5/114.15.2, eBioscience), anti-TSHR (2C11, Santa Cruz Biotechnology), anti-His (9F2, Wako), and anti-Flag (purified IgG, M2, Sigma-Aldrich) Abs were used for flow cytometry. anti-Flag (biotin-conjugated, M2, Sigma-Aldrich) and anti-His (28-75, Wako) Abs were used for immunoprecipitation. anti-Flag (purified IgG, M2, Sigma-Aldrich) and anti-His (9F2, Wako) Abs were used for Western blotting. Anti–HLA-DP (E-20, Santa Cruz Biotechnology) and anti-TSHR (HPA026680, Sigma-Aldrich) polyclonal Abs were used for immunofluorescence staining and PLA. To produce human anti-TSHR mAbs, the V regions of the mAbs were synthesized according to the published sequence (accession numbers: M22 heavy chain, 3G04_B; λ chain, 3G04_A; K1-18 heavy chain, AMF37397; λ chain, AMF37401; K1-70 heavy chain, 2XWT_A; and λ chain, 2XWT_B). For germlined K1-18, some mutants were generated using the QuikChange Multi Mutagenesis Kit (Agilent Technologies) from wild-type K1-18 V regions (heavy chain, N31S, Y54G, and V97A; λ chain, N31S and N32S). These V regions were cloned into pME18S expression vectors containing the secreted form of IgG1 constant region as previously described (14 (link)). HEK293T cells were purchased from RIKEN Cell Bank.
Hek293t cells
Manufactured by RIKEN Cell Bank
Sourced in Japan
HEK293T cells are a widely used human embryonic kidney cell line. They are derived from human embryonic kidney cells transformed with the T antigen of the SV40 virus. HEK293T cells are known for their high transfection efficiency and ability to produce high levels of recombinant proteins.
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Lab products found in correlation
Polyethylenimine (pei), by Merck Group (3 mentions)
Glutamax, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Fujifilm (2 mentions)
Recombinant human basic fgf, by Fujifilm (2 mentions)
Recombinant human egf, by Fujifilm (2 mentions)
Dmem f12, by Fujifilm (2 mentions)
B 27 supplement without vitamin a, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Nacalai Tesque (2 mentions)
Penicillin, by Nacalai Tesque (2 mentions)
Streptomycin, by Nacalai Tesque (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (2 mentions)
Anti mhc class 2 m5 114, by Thermo Fisher Scientific (1 mentions)
Anti his 9f2, by Fujifilm (1 mentions)
Anti hla dp e 20, by Santa Cruz Biotechnology (1 mentions)
Anti tshr hpa026680, by Merck Group (1 mentions)
Quikchange multi mutagenesis kit, by Agilent Technologies (1 mentions)
Anti flag, by Merck Group (1 mentions)
15 protocols using hek293t cells
1
Generation of Human Anti-TSHR Monoclonal Antibodies
2
Generating Fluorescent Nuclear-Labeled LLCPK1 Cells
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LLCPK1 cells were cultured in DMEM (low glucose, Wako) containing 10% fetal bovine serum (FBS, Serum Source International). H2B-mCherry- or H2B-Azurite-expressing monoclonal LLCPK1 cells were first generated using the lentivirus system. Probe-expressing, nuclei-labeled LLCPK1 cells were generated with lentivirus infections. HEK293T cells (RIKEN Cell Bank) were cultured in DMEM (high glucose, Wako) medium containing 10% FBS. All cell lines were cultured at 37 °C under 5% CO2.
3
Culturing and Transfecting HEK293T Cells
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Human embryonic kidney HEK293T cells (RIKEN Cell Bank) were maintained in 10% (v/v) fetal bovine serum (FBS)‐containing Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 μg/ml penicillin‐streptomycin solution. Cells were cultured at 37°C under 5% CO2 in humidified conditions.
Transfection of HEK293T cells was performed as follows: cells were plated in a cell culture dish or multi‐well plate and cultured overnight. The next day, the cells were transfected using 25‐kDa branched polyethyleneimine (PEI, Sigma). The ratio of plasmid DNA to PEI was 1: 4 (weight). After 24–96 h, the cells were used in the subsequent experiments. Cell culture supernatant was collected after 2–4 days and centrifuged at 1500 ×g for 5 min to remove cell debris.
Transfection of HEK293T cells was performed as follows: cells were plated in a cell culture dish or multi‐well plate and cultured overnight. The next day, the cells were transfected using 25‐kDa branched polyethyleneimine (PEI, Sigma). The ratio of plasmid DNA to PEI was 1: 4 (weight). After 24–96 h, the cells were used in the subsequent experiments. Cell culture supernatant was collected after 2–4 days and centrifuged at 1500 ×g for 5 min to remove cell debris.
4
Culturing Human Cell Lines
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The HEK293T cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured in Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100U/ml penicillin and 100U/ml streptomycin. Immortalized human fibroblasts (hTERT-BJ1) were cultured in DMEM-medium 199 (4:1) supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, and 100 μg/ml streptomycin.
5
Culturing and Differentiating Osteosarcoma Stem Cells
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HEK293T cells were obtained from the RIKEN Cell Bank (Saitama, Japan) and cultured in DMEM (FUJIFILM Wako Pure Chemical) supplemented with 10% FBS (Hyclone) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO2 (42 (link)). The patient-derived OS cell line 143B was obtained from the ATCC (Manassas, USA) and cultured in adherent medium containing DMEM supplemented with 10% FBS, 110 μg/mL sodium pyruvate (FUJIFILM Wako Pure Chemical), and 1% penicillin/streptomycin. Both cell types were cultured in tissue culture dishes (SARSTEDT) to ensure optimal adherence and expansion. To enrich stem-like cells, 143B cells were harvested using trypsin (BD Bioscience) and EDTA (FUJIFILM Wako Pure Chemical), then cultured in osteosphere medium containing DMEM/F12 (FUJIFILM Wako Pure Chemical) supplemented with 20 ng/mL recombinant human EGF (FUJIFILM Wako Pure Chemical), 20 ng/mL recombinant human basic FGF (FUJIFILM Wako Pure Chemical), B27 supplement without vitamin A (Gibco), GlutaMAX (Thermo Fisher Scientific), and 1% penicillin/streptomycin. Under these conditions, the cells were incubated in Ultra-Low Attachment Surface culture dishes (Corning). To assess the differentiation potential of OSCs, the cells were transferred from osteosphere to adherent medium, and from Ultra-Low Attachment Surface to tissue culture dishes, to promote adherence and differentiation.
6
Cell Line Maintenance and Culture
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HEK293t cells were obtained from RIKEN Cell Bank (Saitama, Japan). HEK-TVA, HEK-TVB, BHK-T7, BHK-EnvA, BHK-EnvB, and B7GG cells were gifted by Dr. Callaway (Salk Institute for Biological Studies, La Jolla, CA, USA; Wickersham et al., 2007 (link); Choi et al., 2010 (link); Osakada et al., 2011 (link); Osakada and Callaway, 2013 (link)). These cells were maintained in DMEM (Wako, Osaka, Japan), supplemented with 10% fetal bovine serum (FBS; Sigma, St. Louis, MI, USA), 100 U/ml penicillin G, and 100 μg/ml streptomycin (Wako). Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C.
7
Cell Culture and Transfection Protocols
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HEK293T cells (RIKEN Cell Bank, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (COSMO Bio, Brazil) at 37 °C in a humidified incubator under an atmosphere containing 5% CO2. Unless otherwise noted, cells were plated in 48-well plates at 6 × 104 cells/well; cell counts were determined using a hemocytometer. The next day, cells were transfected with the indicated vectors using PEI reagent. H3122 and H2228 cells were kindly provided by Dr. Jeffrey A Engelman (Novartis Institutes for Biomedical Research, Cambridge, MA, USA) [35 (link),36 (link)]. Both cells were cultured in RPMI 1640 (Nacalai Tesque) medium supplemented with 10% FBS and transfected using the Lipofectamine 3000 Reagent (Invitrogen, Carlsbad, CA, USA).
8
Glioblastoma Stem Cell Culture Protocol
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HEK293T cells and HEK293GP cells were purchased from the RIKEN Cell Bank and Takara Bio, respectively. These cells were cultured at 37°C in a 5% CO2 incubator and maintained in DMEM supplemented with FBS. Human patient-derived GBM cell lines TGS-01 and TGS-04 were established as described previously (23 (link)). The use of these human materials and protocols were approved by the Ethics Committees of Gifu Pharmaceutical University (Gifu, Japan) and the University of Tokyo (Tokyo, Japan). These cells were confirmed as GSCs and cultured in neurosphere medium containing DMEM/F12 (FUJIFILM Wako Pure Chemical) supplemented with recombinant human EGF at 20 ng/mL (FUJIFILM Wako Pure Chemical), recombinant human basic FGF at 20 ng/mL (FUJIFILM Wako Pure Chemical), B27 supplement without vitamin A (Gibco), and GlutaMAX (Gibco).
9
Cell Culture Protocols for SARS-CoV-2 Research
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HEK293T cells (RIKEN Cell Bank), Huh7 cells (the National Institute of Infectious Diseases) and TMPRSS2-expressing VeroE6 cells (Japanese Collection of Research Bioresources Cell Bank, JCRB1819) were cultured in DMEM (Nacalai, Japan) supplemented with 10% FBS (Biological Industries, USA), penicillin (100 U/mL), and streptomycin (100 μg/mL) (Nacalai, Japan) and cultured at 37°C in 5% CO2. The Expi293 cells (Thermo) were cultured with the Expi293 medium. The cells were routinely checked for mycoplasma contamination.
10
HEK293T Cell Authentication
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HEK293T cells were obtained from the RIKEN Cell Bank. Cell lines were authenticated by the provider and routinely tested for mycoplasma infection.
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