HeLa cells (MilliporeSigma) were transfected with either a CMV-driven MAFA or MAFB expression plasmid using the Lipofectamine protocol (Thermo Fisher Scientific). Forty-eight hours later, nuclear extract and DNA binding reactions were performed. Briefly, 10 μg of nuclear extract and 200 fmol of the biotin-labeled double-stranded human INS MAFA/B binding site probe were mixed either alone or with unlabeled competitor DNAs in a 20 μL reaction system (LightShift Chemiluminescent EMSA Kit, Thermo Fisher Scientific) containing 1× binding buffer, 2.5% glycerol, 5 mM MgCl, 50 ng/μL of poly(dI-dC) (Thermo Fisher Scientific), and 0.05% NP-40 (Sigma). MAFA and MAFB antibody super-shift reactions were performed. The WT and mutant MAFA/B binding site sequences are provided in Supplemental Table 6 . The reactions were separated on a 6% precast DNA retardation gel in 0.5% Tris borate-EDTA buffer (TBE, Thermo Fisher Scientific) at 100 V for 1.5 hours.
Hela cells
Manufactured by Merck Group
Sourced in United States, United Kingdom, France, Brazil
HeLa cells are a type of immortalized human cell line derived from cervical cancer cells. They are commonly used in biomedical research as a model system for various studies, including cell biology, genetics, and drug development. HeLa cells have the ability to continuously divide and proliferate, making them a valuable tool for researchers.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
L glutamine, by Merck Group (6 mentions)
Penicillin, by Thermo Fisher Scientific (5 mentions)
Streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (4 mentions)
Penicillin streptomycin, by Merck Group (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Hela cell, by American Type Culture Collection (4 mentions)
Thymidine, by Merck Group (3 mentions)
Fetal calf serum (fcs), by Merck Group (3 mentions)
Nocodazole, by Merck Group (3 mentions)
Trypsin edta, by Merck Group (3 mentions)
Hela cell, by Thermo Fisher Scientific (3 mentions)
Lncap, by Merck Group (2 mentions)
Ovcar3, by Merck Group (2 mentions)
Authenticated cancer cell lines, by Merck Group (2 mentions)
Infinite m1000 pro plate reader, by Tecan (2 mentions)
Dmem medium, by Merck Group (2 mentions)
52 protocols using hela cells
1
Quantification of MAFA/B DNA Binding
2
Developing Novel Proteasome Inhibitors for Cancer Treatment
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Example 8
New proteasome inhibitors may be developed not only for treating conditions mediated by senescent cells, but also conditions mediated by cancer cells.
The ability of compounds to specifically kill cancer cells can be tested in assays using other established cell lines. These include HeLa cells, OVCAR-3, LNCaP, and any of the Authenticated Cancer Cell Lines available from Millipore Sigma, Burlington Mass., U.S.A. Compounds specifically kill cancer cells if they are lethal to the cells at a concentration that is at least 5-fold lower, and preferably 25- or 100-fold lower than a non-cancerous cell of the same tissue type. The control cell has morphologic features and cell surface markers similar to the cancer cell line being tested, but without signs of cancer.
In vivo, compounds are evaluated in flank xenograft models established from sensitive SCLC (H889) and hematologic (RS4; 11) cell lines, or using other tumor-forming cancer cell lines, according to what type of cancer is of particular interest to the user. When dosed orally or intravenously, compounds induce rapid and complete tumor responses (CR) that are durable for several weeks after the end of treatment in all animals bearing H889 (SCLC) or RS4; 11 (ALL) tumors. Similar treatment of mice bearing H146 SCLC tumors can induce rapid regressions in the animals.
3
Evaluating Novel Proteasome Inhibitors for Cancer Treatment
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Example 8
New proteasome inhibitors may be developed not only for treating conditions mediated by senescent cells, but also conditions mediated by cancer cells.
The ability of compounds to specifically kill cancer cells can be tested in assays using other established cell lines. These include HeLa cells, OVCAR-3, LNCaP, and any of the Authenticated Cancer Cell Lines available from Millipore Sigma, Burlington Mass., U.S.A. Compounds specifically kill cancer cells if they are lethal to the cells at a concentration that is at least 5-fold lower, and preferably 25- or 100-fold lower than a non-cancerous cell of the same tissue type. The control cell has morphologic features and cell surface markers similar to the cancer cell line being tested, but without signs of cancer.
In vivo, compounds are evaluated in flank xenograft models established from sensitive SCLC (H889) and hematologic (RS4;11) cell lines, or using other tumor-forming cancer cell lines, according to what type of cancer is of particular interest to the user. When dosed orally or intravenously, compounds induce rapid and complete tumor responses (CR) that are durable for several weeks after the end of treatment in all animals bearing H889 (SCLC) or RS4;11 (ALL) tumors. Similar treatment of mice bearing H146 SCLC tumors can induce rapid regressions in the animals.
4
Cell Culture Conditions for NF-κB Assays
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HeLa cells (Merck) were cultured in MEM Earle’s medium (PAN) supplemented with l -glutamine (2 mM, PAN), non-essential amino acids (1%, PAN), penicillin and streptomycin (1%, PAN) and fetal calf serum (FCS; 10%, PAN) under standard conditions (5% CO2, 37 °C). HEK293T cells (DSMZ) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; PAN) supplemented with l -glutamine (2 mM, PAN), penicillin and streptomycin (1%, PAN) and FCS (10%, PAN) under standard conditions (5% CO2, 37 °C).
HEK-NF-κB cells (TRON) were cultured under standard conditions (5% CO2, 37 °C) in DMEM supplemented with FCS (10%), HEPES buffer (1%),l -glutamine (1%), non-essential amino acids (1%) and sodium pyruvate (1%). For selection, the following antibiotics were added to the culture of the HEK-NF-κB-Null, HEK-NF-κB-TLR7 and HEK-NF-κB-TLR8 cell lines: blasticidin (10 µg ml−1), Zeocin (100 µg ml−1) and Geneticin (G418; 250 µg ml−1). The HEK-NF-ĸB-TLR3 cell line was cultured in the absence of Geneticin. All of the cell lines overexpress an NF-κB driven Firefly luciferase, which allows the detection of NF-κB production in a luminescence assay and can be used as an indicator for the induction of an immune response46 (link). Additionally, the cell lines HEK-NF-κB-TLR3, HEK-NF-κB-TLR7 and HEK-NF-κB-TLR8 overexpress the respective TLRs.
HEK-NF-κB cells (TRON) were cultured under standard conditions (5% CO2, 37 °C) in DMEM supplemented with FCS (10%), HEPES buffer (1%),
5
Cell Culture Protocols for HEK293T and HeLa Cells
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HEK293T cells
(DSMZ) were cultured in Dulbecco’s modified Eagle’s
medium (PAN) enriched with penicillin and streptomycin (1%, PAN),l -glutamine (2 mM, PAN), and fetal calf serum (FCS) (10%, PAN)
under standard conditions (5% CO2, 37 °C). HeLa cells
(Merck) were cultured in MEM Earle’s medium (PAN) enriched
with penicillin and streptomycin (1%, PAN), nonessential amino acids
(1%, PAN),l -glutamine (2 mM, PAN), and FCS (10%, PAN) under
standard conditions (5% CO2, 37 °C).
(DSMZ) were cultured in Dulbecco’s modified Eagle’s
medium (PAN) enriched with penicillin and streptomycin (1%, PAN),
under standard conditions (5% CO2, 37 °C). HeLa cells
(Merck) were cultured in MEM Earle’s medium (PAN) enriched
with penicillin and streptomycin (1%, PAN), nonessential amino acids
(1%, PAN),
standard conditions (5% CO2, 37 °C).
6
HeLa Cell mRNA Transfection and Viability Assay
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HeLa cells (Merck) were cultured as mentioned above. One day before transfection, the cells were seeded in a 96-well plate (30,000 cells per well) and cultured in minimal essential medium (MEM) with antibiotics. The cells were transfected with mRNA (100 ng) in Opti-MEM (10 µl) using Lipofectamine MessengerMAX transfection reagent (0.3 µl) in Opti-MEM (9.7 µl). The cells were incubated with the mRNA/Lipofectamine MessengerMAX mixture for 6 h at 37 °C in a total volume of 100 µl. The samples were irradiated under the indicated conditions. Subsequently, the cell medium with the transfection agent was replaced by fresh medium and the cells were incubated overnight at 37 °C in medium. At 24 h post transfection, MTT solution (16.5 mg MTT in 3.3 ml of PBS) was added to the 96-well plate (12.5 µl per well). After 4 h of incubation at 37 °C, the supernatant was removed and 0.04 M HCl in isopropanol was added to the wells. After incubation for 1.5 h at r.t., 100 µl of the supernatant was placed in a new 96-well plate and absorption at 550 nm was measured using the Tecan Infinite M1000 PRO plate reader.
7
Culturing and Characterizing NF-κB Reporting Cell Lines
8
mRNA Transfection and Cell Viability Assay
9
HeLa Cell Culture and Synchronization
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HeLa cells, from American Tissue Culture Collection, were maintained as subconfluent monolayers in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% (v/v) fetal bovine serum (FBS; Hyclone) and 100 IU/ml penicillin plus 100 mg/ml streptomycin (Gibco) at 37°C with 5% CO2. For cell synchronization, aliquots of HeLa cells were synchronized at G1/S with 2.5 mM thymidine (Sigma-Aldrich) for 16 h, washed with PBS three times, and then cultured in thymidine-free medium for appropriate time intervals.
All the siRNAs or constructs were transfected into HeLa cells with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol.
All the siRNAs or constructs were transfected into HeLa cells with Lipofectamine 2000 (Invitrogen) following the manufacturer’s protocol.
10
Thermal Stress Induction in HeLa Cells
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HeLa cells (originally obtained from RIKEN BRC) were cultured at 37 °C in 5% CO2 in Dulbecco’s modified eagle’s medium (Sigma) containing 10% fetal bovine serum (MP Biomedicals), penicillin and streptomycin (Thermo). For thermal stress induction, the cells were incubated at 42 °C in an incubator with 5% CO2.
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