The colon cancer cell lines (HCT116, COLO320, T24, HT29, SW480, SW948 and SW1417) used in our study were purchased from American Type Culture Collection (ATCC). The colon cancer cell line COLO678 was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). Human HEK293T was purchased from ATCC. The culture medium for SW480 and HEK293T was RPMI-1640 medium (Invitrogen, Carlsbad, California, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 100 U/mL Penicillium and 100 μg/mL Streptomycin. The culture medium for HCT116, COLO320, T24, HT29, SW480, SW948 and SW1417 were DMEM: F12 (1:1) medium (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, Utah, USA), 100 U/mL Penicillium and 100 μg/mL Streptomycin. Cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Replaced the culture medium 2–3 times every week.
Human hek293t
Manufactured by American Type Culture Collection
Sourced in United States
The Human HEK293T is a cell line derived from human embryonic kidney cells. It is a widely used cell line in cell biology and biotechnology research due to its ease of cultivation and high transfection efficiency.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (6 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Hek293t, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Hek293t, by Corning (2 mentions)
Mycoalert mycoplasma detection kit, by Lonza (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Dmem f12 1 1 medium, by Thermo Fisher Scientific (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Hct116, by Thermo Fisher Scientific (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
Sw480, by Thermo Fisher Scientific (1 mentions)
Colo678, by Leibniz Institute DSMZ (1 mentions)
Colo320, by American Type Culture Collection (1 mentions)
Sw1417, by American Type Culture Collection (1 mentions)
Colo320, by Thermo Fisher Scientific (1 mentions)
Sw948, by Thermo Fisher Scientific (1 mentions)
Sw948, by American Type Culture Collection (1 mentions)
18 protocols using human hek293t
1
Culturing Colon Cancer Cell Lines
2
Authenticated Cell Lines for Research
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Human HEK293T (ATCC CRL-3216), Human HEK293S GnTI- (ATCC CRL-3022), Spodoptera frugiperda Sf9 (ATCC CRL-1711). All the cell lines are authenticated and tested by manufacturer for mycoplasma contamination. No additional test was performed by the authors of this study. No commonly misidentified lines were used.
3
Cell Culture Conditions for Diverse Cell Lines
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Baby hamster kidney BHK-21 (American Type Culture Collection, ATCC, CCL-10), house mouse L929 (ATCC CCL-1), grivet Vero E6 (ATCC CRL-1586), human A549 (ATCC CCL-185), and human HEK 293T (ATCC CRL-3216) cells were grown in Dulbecco’s modified Eagle’s medium (Thermo Fisher Scientific, Waltham, MA) containing 10% heat-inactivated fetal bovine serum, 2 mM of L-glutamine, 100 mg/ml of streptomycin, and 100 U/ml of penicillin at 37°C and 5% CO2.
4
Mouse and human cell culture protocol
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Mouse E14 cells and human HEK293T were purchased from ATCC. Mouse E14 cells were cultured in 0.2% gelatine‐treated dish with medium containing Knockout Dulbecco's modified eagle medium (DMEM) (Gibco) and 20% fetal bovine serum (FBS) (Hyclone, Thermo), 1% Penicillin–streptomycin (Gibco), 1% L‐glutamine (Gibco), 1% non‐essential amino acid (Gibco), 0.1 mM β‐mercaptoethanol (Sigma‐Aldrich) and incubated at 37°C and 5% atmospheric CO2 and 100% relative humidity. HEK293T cells were cultured in DMEM (Corning) and 10% FBS (Lonsera). Polyethylenimine, linear (PEI) was used for transfection in HEK293T, while transfection in E14 cells was performed using LipoFectMax 3000 (ABP biosciences, FP319M). Cells were collected 3 days after transfection for further analysis.
5
Validated HEK293T Cell Culture Protocol
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Human HEK293T (Cat# CRL-11268 from ATCC; RRID: CVCL_QW54) was obtained and cultured in DMEM (GIBCO) with 10% FBS (GIBCO) in 5% CO2 at 37 °C. Cells validation using short tandem repeat markers (STR) were performed by Meixuan Biological Science and Technology Ltd. (Shanghai). In detail, these cell lines were firstly tested cell species by PCR method using extracted total genomic DNA, and examined by STR profiling. Then, STR data were analyzed using the DSMZ (German Collection of Microorganisms and Cell Cultures) online STR database (http://www.dsmz.de/fp/cgi-bin/str.html ). Cell lines were tested negative for mycoplasma contamination.
6
Cell Culture of Diverse Lines
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Human HEK293A, human HEK293T and murine Raw264.7 cell lines were obtained from ATCC (Manassas, US). Cells were cultured in DMEM containing 10% FBS at 5% CO2 and 37 °C.
7
Cell Line Authentication and Validation
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Human HEK293T (Cat# CRL-11268 from ATCC; RRID: CVCL_QW54), MKN45 (Cat# JCRB0254 from Japanese Collection of Research Bioresources (JCRB) Cell Bank, RRID: CVCL_0434), MGC803 (Cat# C6582 from Beyotime Biotechnology, RRID: CVCL_5334), and NCI-N87 (Cat# CRL-5822 from ATCC; RRID: CVCL_1603), were obtained and cultured in DMEM (GIBCO) with 10% FBS (GIBCO) in 5% CO2 at 37 °C. Cells validation using short tandem repeat markers (STR) were performed by Meixuan Biological Science and Technology Ltd. (Shanghai). In detail, these cell lines were firstly tested cell species by PCR method using extracted total genomic DNA, and examined by STR profiling. Then, STR data were analyzed using the DSMZ (German Collection of Microorganisms and Cell Cultures) online STR database (http://www.dsmz.de/fp/cgi-bin/str.html ). Cell lines were tested negative for mycoplasma contamination. All cells were grown according to the instruction.
8
Cell Culture Protocol for HEK 293T and HCT116
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Human HEK 293T (ATCC, Manassas, VA) and
HCT116 (a generous gift from Dr. Fred Dick at the University of Western
Ontario) cell lines were cultured in Dulbecco’s modified Eagle’s
medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum
(Wisent, Quebec, Canada) and 1% penicillin-streptomycin (HyClone).
All cell lines were grown at 37 °C in the presence of 5% CO2 and subcultured following the supplier’s recommendations.
Cultured cells were routinely screened with the MycoAlert Mycoplasma
Detection Kit (Lonza, Basel, Switzerland).
HCT116 (a generous gift from Dr. Fred Dick at the University of Western
Ontario) cell lines were cultured in Dulbecco’s modified Eagle’s
medium (HyClone, Logan, UT) supplemented with 10% fetal bovine serum
(Wisent, Quebec, Canada) and 1% penicillin-streptomycin (HyClone).
All cell lines were grown at 37 °C in the presence of 5% CO2 and subcultured following the supplier’s recommendations.
Cultured cells were routinely screened with the MycoAlert Mycoplasma
Detection Kit (Lonza, Basel, Switzerland).
9
EV Isolation and Characterization from HEK293T
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Human HEK293T (ATCC, Manassas, VA, USA) were maintained in a humidified incubator at 5% CO 2 . HEK293T cells were cultured in high glucose DMEM (Corning, New York, NY, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 5% penicillin and streptomycin (Thermo Fisher Scientific, Waltham, MA). Cells were cultured at 70-80% confluency in a 6-well plate (Thermo Fisher Scientific, Waltham, MA) and transiently transfected with CD63-pEGFP C2 (#62964) gag-GFP (#80605) and pEGFP-C1 (#54759) constructs purchased from Addgene. To collect cell-derived EVs, cells were cultured in serum-free media for 48 hours. 55 Ti rotor to deplete EVs. After the process of sequential centrifugation, plasma samples were diluted in DPBS. 10 μL of diluted plasma samples were incubated with anti-PSMA and STEAP1 antibodies in dark at room temperature for 30 minutes followed by adding 180 μL of DPBS to the mixture. The prepared sample was run with A60MP.
10
Establishment of Gemcitabine-Resistant CCA Cell Lines
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CCA cell lines HuCCT1 and HuH28 were purchased from Japan Health Science Research Resources Bank, and CCA cell lines SNU-1196, SNU-1079, SNU-308, SNU-245, SNU-478 and SNU-869 were purchased from Korea Cell Line Bank. Human HEK293T was obtained from American Type Culture Collection (ATCC). All cell lines were cultured with RPMI-1640 medium (Invitrogen, USA) supplemented with 10% fetal bovine serum (Hyclone, USA) and 1% PenStrep (100 U/mL Penicilium and 100 μg/mL Streptomycin) in a humid atmosphere containing 5% CO2 at 37 °C. Gemcitabine resistant cell lines HuCCT1-Gem and SNU-245-Gem were constructed by exposing to increasing dosages of Gemcitabine (Selleck Chemicals, USA) for 8 months, and then persistently cultured in medium containing 10 nmol/l Gemcitabine.
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