HeLa and HEK293T, were purchased from ATCC (without further authentication) and cultured in DMEM GlutaMAXTM medium supplied with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin (10,000 U/mL). Cell lines used in this paper are not listed in the database of commonly misidentified cell lines maintained by ICLAC. Mouse embryonic fibroblasts (MEFs) were obtained as previously described (35 (link)). Fibroblast from patients were obtained and cultured as previously described (20). HLE-B3 cells were purchased from ATCC and cultured in MEM-a supplied with 20% fetal bovine serum (FBS), 1% Penicillin-Streptomycin (10,000 U/mL) and GlutaMAX. Cell lines were routinely tested formycoplasma contamination.
Hle b3 cells
Manufactured by American Type Culture Collection
Sourced in United States
The HLE-B3 cells are a type of human lens epithelial cell line. They are derived from the lens epithelium of a human eye and are commonly used in vision research and cell biology studies.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Antimicrobial solution, by Merck Group (1 mentions)
Epigallocatechin gallate (egcg), by Merck Group (1 mentions)
Hyclone, by Cytiva (1 mentions)
Hle b3, by Thermo Fisher Scientific (1 mentions)
Hle b3, by Merck Group (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Ly294002, by Cell Signaling Technology (1 mentions)
Recombinant human tgf β2, by Thermo Fisher Scientific (1 mentions)
Tgfβ2, by Cell Signaling Technology (1 mentions)
Arpe 19 cell, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Sh3039603, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
15 protocols using hle b3 cells
1
Cell Line Cultivation and Authentication
2
Cell Culture Protocol for HLEB3 and HEK293T
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The HLEB3 cells (ATCC CRL-11421) were ordered from ATCC. HEK293T cells were obtained from the Laboratory cell bank of Shenzhen PKU-HKUST Medical Center, which were initially ordered from ATCC by colleagues in HKUST. Cell culture was performed according to the instructions (Andley et al., 1994 (link)). The mycoplasma contamination was prevented by examining the integrity of cell nucleus via DAPI staining.
3
Hyperosmolar Stress on HLE-B3 Cells
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HLE-B3 cells, a HLEC line immortalized by viral transformation of SV-40, were purchased from the American Type Culture Collection (Manassas, VA, USA). These cells were maintained in Minimum Essential Medium (MEM; Cat # 11095080; Lot No. 2120598; Gibco, USA) containing 10% fetal bovine serum and 1% antimicrobial solution (Sigma-Aldrich, St. Louis, MO, USA) in 5% CO2 at 37 °C. In the entire experiment, when only media were used, they were described as normal media (NM), and hyperosmolar stress was applied by adding 50, 100, and 150 mOsm/L of NaCl to this baseline media. The osmolarity of the media itself could have a very important effect on this study, so we measured the osmolarity of the normal media using a 2430 Multi-Osmette auto-sampling turntable osmometer (Precision System Inc., Natick, MA, USA). As a result, the osmolarity of the normal media was measured as 291.5 ± 1.0 mOsm/L (n = 4). All cultures were grown to ~80–90% confluency prior to experiments. To induce hyperosmotic stress, sterile sodium chloride (1 M) was added to the culture media. Cells were incubated in hyperosmolar medium for 24, 48, or 72 h. An osmolarity range of 300–450 mOsm/L was selected based on previous data indicating that osmolarity in areas of tear breakup can reach up to 560 mOsm/L in the precorneal tear layer [26 (link)].
4
Hypoxia-Induced Oxidative Stress Assay
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HLE-B3 cells purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) were grown as a monolayer in DMEM supplemented with 20% heat-inactivated fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2 and 21% O2. Twenty-four h before the day of the experiment, cells were switched to hypoxic conditions (1% O2 to mock physiological environment [15 (link)]). At 85–90% confluence, the cells were treated with the indicated concentration of H2O2 for 24 h.
5
UVB-Induced Stress Response in HLE B-3 Cells
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HLE B-3 cells were obtained from the American Type Culture Collection (ATCC); this cell line was authenticated by STR. The cells were cultured in RPMI 1640 medium (HyClone; Cytiva) containing 10% fetal bovine serum (HyClone; Cytiva) at 37°C in a humidified environment containing 5% CO2. After reaching 75–80% confluence, the cells were irradiated with 30 mJ/cm2 UVB at room temperature for 2 min or pretreated with EGCG (MilliporeSigma) for 2 h prior to UVB irradiation at 37°C. At the designated time points, the cells were collected for different measurements.
6
In vitro Cataract Model using H2O2-induced Apoptosis
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HLE-B3 cells were obtained from the American Type Culture Collection. HLE-B3 cells were cultured in minimum essential medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin, and maintained in a humidified incubator with 5% CO2 at 37˚C.
Oxidants induce cell apoptosis and trigger the development of cataracts (10 (link)). As peroxidative damage is mediated by the toxic metabolites of oxygen, such as hydroxide, H2O2 is frequently used to induce the apoptosis of LECs in vitro. For the establishment of an in vitro cataract model, HLE-B3 cells (1x106 cells/well) were seeded into 6-well plates and induced at 37˚C with 200 µmol/l H2O2 (Sigma-Aldrich; Merck KGaA) for 24 h, as previously described (17 (link),18 (link)), while cells in control group were untreated.
Oxidants induce cell apoptosis and trigger the development of cataracts (10 (link)). As peroxidative damage is mediated by the toxic metabolites of oxygen, such as hydroxide, H2O2 is frequently used to induce the apoptosis of LECs in vitro. For the establishment of an in vitro cataract model, HLE-B3 cells (1x106 cells/well) were seeded into 6-well plates and induced at 37˚C with 200 µmol/l H2O2 (Sigma-Aldrich; Merck KGaA) for 24 h, as previously described (17 (link),18 (link)), while cells in control group were untreated.
7
Culturing HLE B-3 Cells in MEM
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HLE B-3 cells were obtained from American Type Culture Collection (ATCC; Rockville, MD, USA) and cultured in Eagle’s minimum essential medium (Gibco-BRL, Grand Island, NY, USA) with 20 % FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified 5 % CO2 atmosphere.
8
Oxidative Stress and Senescence in HLE-B3 Cells
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HLE-B3 cells (American Type Culture Collection, Manassas, VA, USA) were obtained from the Department of Ophthalmology, Eye and ENT Hospital of Fudan University. The HLE-B3 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Hyclone, GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 20% fetal bovine serum (FBS; Gibco, South America), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco, USA) in a humidified 5% CO2 atmosphere at 37°C. The medium was changed every 3 days. Cells were detached from culture flask using trypsin (Gibco, USA), counted, seeded in 6-well plates and incubated overnight. Then, the cells were treated with 0, 50, 75, 100, 150, or 200 μM H2O2 for different numbers of days. To study senescence, the cells treated with 150 μM H2O2 were incubated with Met (Sigma-Aldrich) at different concentrations (0.5, 1.0, 2.0 mM) for 7 days. The incubation without Met was used as control.
9
Culturing HLE B3 Cells in DMEM
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HLE B3 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), grown in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Beijing, China) supplemented with 15% premium fetal bovine serum (FBS) (Biological Industries, Israel), 50 U/ml of penicillin and 50 μg/ml streptomycin (Hyclone, Beijing, China). Cells were maintained at 37°C in a humidified 5% CO2 atmosphere.
10
Immortalized HLEB-3 Cell EMT Induction
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Immortalized HLEB-3 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), placed in a humidified atmosphere containing 5% CO2 at 37°C. The cells were washed with phosphate-buffered saline (PBS), and dissociated with 0.05% trypsin and 0.02% EDTA. As in our previous experiments (7 (link)), when the cell cultures reached a confluence of 80%, the cells were divided into three groups (1×106 cells/ml each). In the first group, the cells were stimulated with 10 ng/ml recombinant human TGF-β2 (Peprotech, Inc., Rocky Hill, NJ, USA) for 24 h in serum-free medium to induce EMT. This was termed the TGF-β2 group. To determine the effect of PI3K inhibition on EMT, the cells were pretreated with LY294002 (Cell Signaling Technology, Inc., Danvers, MA, USA) at an appropriate concentration for 1 h prior to being co-treated with 10 ng/ml TGF-β2 for 24 h. This was termed the LY294002+TGF-β2 group. The control group consisted of cells, which were incubated under conventional conditions without the presence of either TGF-β2 or LY294002 in the medium. Following treatment, the cells were collected for western blot analysis and confocal immunofluorescence assays.
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