Cell lines LNCaP (LNCaP-FGC, RRID:CVCL_1379), CWR22Rv1 (22Rv1, RRID:CVCL_1045), VCaP (RRID:CVCL_2235), DU145 (RRID:CVCL_0105), PC3 (RRID:CVCL_0035) and HEK293T (293T, RRID:CVCL_0063) were purchased from ATCC, PNT2 was purchased from Sigma–Aldrich (Cat# 95012613, RRID:CVCL_2164). All cell lines were cultured under conditions as recommended. C4-2B and C4-2B/EnzR cells were generous gifts from Dr. Allen C. Gao at UC Davis. Parental C4-2B cells were cultured in the RPMI-1640 medium supplemented with 10% FBS, while C4-2B/EnzR cells were maintained in the RPMI-1640 complete medium containing 10 μM enzalutamide. All cell lines were recently validated with STR analysis, and confirmed to be mycoplasma-free by Mycoplasma PCR Detection Kit (GeneCopoeia, Rockville, MD USA). Enzalutamide (Enz) was purchased from Selleck Chemicals (Houston, TX, USA).
Hek293t 293t
Manufactured by American Type Culture Collection
HEK293T (293T) is a widely used human embryonic kidney cell line. It is derived from the HEK293 cell line and is known for its high transfection efficiency and robust growth characteristics. The 293T cell line expresses the SV40 large T antigen, which allows for high-level expression of transfected genes.
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Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hgc 27, by American Type Culture Collection (2 mentions)
Mkn45, by American Type Culture Collection (2 mentions)
Nci n87, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Lncap lncap fgc, by American Type Culture Collection (1 mentions)
Cwr 22rv1 22rv1, by American Type Culture Collection (1 mentions)
Mycoplasma pcr detection kit, by GeneCopoeia (1 mentions)
Enzalutamide enz, by Selleck Chemicals (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Anti vsv g antibody i1 mouse hybridoma supernatant, by American Type Culture Collection (1 mentions)
1640 medium, by Nacalai Tesque (1 mentions)
Penicillin, by Nacalai Tesque (1 mentions)
Streptomycin, by Nacalai Tesque (1 mentions)
Amphotericin b, by Nacalai Tesque (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Dld 1, by American Type Culture Collection (1 mentions)
Ncm460, by American Type Culture Collection (1 mentions)
10 protocols using hek293t 293t
1
Prostate Cancer Cell Line Characterization
2
Production of SARS-CoV-2 Spike Pseudoviruses
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The BtKY72 S construct was synthesized by GenScript and cloned into an HDM plasmid with a C-terminal 3X FLAG tag. The BtKY72 mutant S constructs T498W (BtKY72 S residue 487) and K493Y/T498W (BtKY72 S residue 482/487) were subcloned by GenScript from the BtKY72 S construct. Pseudotyped VSV particles were prepared using HEK293T (293T) (ATCC CRL-11268) cells seeded into 10-cm dishes. 293T cells were transfected using Lipofectamine 2000 (Life Technologies) with a S encoding-plasmid in Opti-MEM transfection medium and incubated for 5 hr at 37°C with 8% CO2 supplemented with DMEM containing 10% FBS. One day post-transfection, cells were infected with VSV (G*ΔGluciferase), and after 2 hr, infected cells were washed 5x with DMEM before adding medium supplemented with anti-VSV G antibody (I1-mouse hybridoma supernatant diluted 1:40, from ATCC CRL-2700). Pseudotyped particles were harvested 18-24 hr post-inoculation, clarified from cellular debris by centrifugation at 3000 g for 10 min, concentrated 100x using a 100 MWCO membrane for 10 min at 3000 rpm, and frozen at -80°C. Mock pseudotyped VSV pseudovirus was generated as above but in the absence of S.
3
SARS-CoV-2 nsp12 Antibody Development
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P3X63Ag8U.1 (P3U1) and SV40-transformed human embryonic kidney cell line HEK-293T (293T) cells were obtained from the American Type Culture Collection (Manassas, VA). P3U1 cells were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Nacalai Tesque, Inc., Kyoto, Japan) that was supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA), 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.). 293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS, streptomycin (100 μg/ml), and penicillin (100 U/ml).
Anti-β-actin (AC-15) mouse mAb (Merck, Darmstadt, Germany) and anti-FLAG mouse mAb (M2) (Merck) were used for western blotting analysis. mAbs against nsp12 of SARS-CoV-2 (RdMab-2, -13, and -20) were developed in this study (see below).
Anti-β-actin (AC-15) mouse mAb (Merck, Darmstadt, Germany) and anti-FLAG mouse mAb (M2) (Merck) were used for western blotting analysis. mAbs against nsp12 of SARS-CoV-2 (RdMab-2, -13, and -20) were developed in this study (see below).
4
Characterization of CRC Cell Lines
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Human CRC cell lines (HCT8, DLD1, HCT116 and HT29), normal colorectal epithelial cell lines (NCM460 and CCC-HIE-2) and HEK-293T (293T) cells were purchased from the American Type Culture Collection (ATCC). All of these cell lines were cultured according to the instructions recommended by ATCC, and characterized by Genewiz (China) using short tandem repeat markers.
5
Cell Line Characterization and Manipulation
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U87, A172, and HEK293T (293T) cells were purchased from American Type Culture Collection. HeLa cells stably expressing empty vector (EV), 6His-SUMO 1 and 6His-SUMO 2, were obtained from Dr. RT Hay and have been described previously [37 (link)]. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (ThermoFisher Scientific). GBM34 and GBM44 GSCs obtained from Dr. Mariano Viapiano (Brigham and Women’s Hospital, Boston, MA) were maintained as neurospheres as described [27 ]. All cell lines were screened for mycoplasma using the ATCC Universal Mycoplasma Detection Kit (catalogue # 30- 121 1012K) every 4 months. Early passage primary MEFs from wild-type, MyD88−/−, Rig-I−/−, Mda5−/−, Lgp2−/−, MAVS−/−, Tmem173−/−, and Trif−/− mice were cultured as previously described [51 (link), 52 (link)]. All cell lines were authenticated by routine morphological and growth analysis and by western blotting. TMZ was obtained from Sigma-Aldrich. DNA transfection was performed using TransIT LT1 (Mirus) and siRNA transfection with Oligofectamine (Invitrogen). Cycloheximide (CHX) and MG132 were from Cayman Chemical. The following siRNAs were used: siGENOME non-targeting siRNA#3 (Dharmacon), siGENOME Smartpool si-LGP2 (Dharmacon), si-TLR3 (sc-36685, Santa Cruz), and si-PKR (sc-36263, Santa Cruz).
6
Culturing GC and HEK293T Cell Lines
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GC cell lines (HGC‐27, NCI‐N87, MKN45, and AGS) and HEK293T (293T) were purchased from the American Type Culture Collection (ATCC). HGC‐27 and HEK293T cells were cultured in DMEM, whereas other cells were incubated in RPMI 1640 medium. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
7
Culturing GC and HEK293T Cell Lines
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GC cell lines (HGC‐27, NCI‐N87, MKN45, and AGS) and HEK293T (293T) were purchased from the American Type Culture Collection (ATCC). HGC‐27 and HEK293T cells were cultured in DMEM, whereas other cells were incubated in RPMI 1640 medium. All cells were maintained at 37°C in a humidified atmosphere of 5% CO2.
8
Transfection of piRNA and Plasmid in Cell Lines
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HEK293T (293T) and GC-2spd(ts) cells were obtained from the American Type Culture Collection (ATCC) and cultured with the medium and serum as ATCC recommended. Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. For transfection of the RNA oligonucleotides, 100∼200 nM of piRNA or Scr siRNA oligonucleotides were used. For plasmid, 4 μg DNA was used in a six-well plate. All chemically synthetic piRNAs used for cell culture transfection are 2′-O-methyl modified at their 3′ ends.
9
Characterization of H7 Influenza Viruses
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HEK293T (293T) and Madin-Darby canine kidney (MDCK) cells were obtained from the American Type Culture Collection (ATCC, #CRL-11268 for 293T, female; #PTA-6500 for MDCK, female) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in 37°C, 5% CO2 atmosphere. The H7N9 virus used in the animal study was A/Shanghai/4664T/2013 was obtained from Shanghai Public Health Clinical Center and stored at -80°C in a BSL-3 laboratory (Fudan University). A/Shanghai/4664T/2013 has very similar sequence to that of A/Shanghai/2/2013 with only a single point mutation (Y282H). The H7N3 A/RT/NJ65/85 and H7N9 A/Shanghai/2/2013 viruses used in the neutralization assay were vaccine strain viruses generated by reverse genetics obtained from CDC. The H7N9 virus titer was expressed as 50% tissue culture infection dose (TCID50). In brief, the virus was serially diluted 10-fold. The diluted virus samples were then added to MDCK cells in 96-well plates, which were incubated at 37°C for 3 days. The TCID50 values were measured by determining CPE and were calculated by the Reed-Muench method. The H7N4 virus used in this study was A/EM/Korea/W266/2007, which was generously provided by Dr. Young-Ki Choi at Chungbuk National University, Republic of Korea.
10
Cell Culture and Viral Strain
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MDCK and HEK293T (293T; American Type Culture Collection) cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U ml–1 penicillin and 100 mg ml–1 streptomycin at 37°C with 5% CO2. The H7N9 strain A/Anhui/1/2013 was used in this study.
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