HaCaT cells (spontaneously transformed human keratinocytes) were obtained from Cell Lines Service (CLS; Eppelheim, Germany). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, France) supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 IU/mL penicillin, and 50 IU/mL streptomycin (Gibco) at 5% CO and 37 C. When the HaCaT cells reached confluency, they were dispersed using trypsin and counted using a hematimeter. The cell suspension was diluted to a cell density of 100,000 cells/mL and seeded in 96-well microplates for 24 h. The compounds in ethanol were prepared at 1:100 dilution in culture medium and incubated for 24 h. Epigallocatechin gallate (EGCG) at 10 µg/mL was used as an anti-inflammatory control [21 (link)].
Hacat cells
Manufactured by Thermo Fisher Scientific
Sourced in United States
HaCaT cells are a spontaneously immortalized human keratinocyte cell line. They are derived from normal human skin and are commonly used as an in vitro model for keratinocyte biology and skin-related research.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Hacat cells, by American Type Culture Collection (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Hs27 cells, by American Type Culture Collection (2 mentions)
Transcriptor first strand cdna synthesis kit, by Roche (2 mentions)
Lightcycler 480, by Roche (2 mentions)
Ez pcr mycoplasma test kit, by Sartorius (2 mentions)
Hacat cell, by Cytion (1 mentions)
Hacat cells, by CLS Cell Lines Service (1 mentions)
Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions)
Rhtnf α, by R&D Systems (1 mentions)
Recombinant human il 17a, by R&D Systems (1 mentions)
Rhil 22, by R&D Systems (1 mentions)
Rhil 1α, by R&D Systems (1 mentions)
Hacat cell, by National Collection of Authenticated Cell Cultures (1 mentions)
Penicillin streptomycin antibiotic, by Thermo Fisher Scientific (1 mentions)
22 protocols using hacat cells
1
Anti-inflammatory Evaluation of EGCG in HaCaT Cells
2
Radiation-Induced DNA Damage in HaCaT Cells
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The human immortalized keratinocytes, HaCaT cells, were obtained from the American Type Culture Collection (ATCC, Manassas, VA). HaCaT cells were maintained in high glucose Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY), supplemented with 10% fetal bovine serum and 100 U/mL penicillin-streptomycin (Gibco, Paisley, PA) at 37°C with 5% CO2 under humidified conditions.
Cells were pre-treated with 10 mM of NAC for a 1 hour before radiation and were irradiated for 10 minutes with 6 MV LINAC (21EX, Varian Medical Systems) at a fixed dose rate of 2 Gy/min. A radiation of 20 Gy was selected to induce DNA damage [12 (link),13 (link)].
Cells were pre-treated with 10 mM of NAC for a 1 hour before radiation and were irradiated for 10 minutes with 6 MV LINAC (21EX, Varian Medical Systems) at a fixed dose rate of 2 Gy/min. A radiation of 20 Gy was selected to induce DNA damage [12 (link),13 (link)].
3
Culturing HaCaT and Hs27 Cell Lines
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HaCaT cells were obtained from CLS Cell Lines Service GmbH (Cat # 300493), and Hs27 cells were purchased from ATCC, American Type Culture Collection (CRL-1634). Dulbecco’s Modified Eagle’s Medium (DMEM) was purchased from Gibco (Cat # 11996-065) and ATCC (Cat # 30-2002). HaCaT cells were grown in DMEM (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Cat # 16000-044, Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin (Cat # P4458, Gibco) in 5% CO2 at 37.5 °C. Hs27 cells were grown in DMEM (ATCC) with 10% fetal bovine in the same conditions. The cells were removed from the wells with 0.25% trypsin EDTA for each passage.
4
HaCaT Cell Cytokine Stimulation and GSDME Knockdown
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Human keratinocyte cell line, HaCaT cells, were obtained from China Center for Type Culture Collection (Wuhan, China). HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) (both from Gibco, CA, USA) in 5% CO2 environment at 37 °C. HaCaT cells were treated with 10 ng/mL recombinant human (rh) IL-17A (R&D, Minneapolis, USA), 10 ng/mL rh OSM (R&D), 10 ng/mL rh TNF-α (R&D), 10 ng/mL rh IL22 (R&D), and 10 ng/mL rh IL1-α (R&D) in combination for 6, 12, 24, or 48 h. Transfection was performed according to previously described methods (45). For GSDME knockdown, HaCaT cells were transfected with pGLVH1/GFP + Puro lentivirus vector containing NC shRNA (shNC sequence: 5’-TTCTCCGAACGTGTCACGT-3’) or GSDME shRNA (shGSDME sequence: 5’-GCAGAAGTGTGTGATCTCTGA-3’) (GenePharma, Shanghai, China). Stable knockdown HaCaT cells were obtained and selected by puromycin incubation.
5
Anti-inflammatory Effects of AA on HaCaT Cells
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AA was obtained from Sigma-Aldrich (St. Louis, MO, USA). Human keratinocyte (HaCaT) cells (ATCC; Manassas, VA, USA) were incubated at 37 °C under 5% CO2 in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Hyclone; Logan, UT, USA) and 1% penicillin-streptomycin antibiotic (GIBCO; Grand Island, NY, USA).
HaCaT cells (1 × 105 cells/mL) were pretreated with AA (5, 10, and 20 μg/mL) for 1 h. Then, they were treated with 0.2 ng/mL IFN-γ and TNF-α for 24 h to induce inflammation. To determine the cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide assay (Promega, Madison, WI, USA) was used [15 (link)].
HaCaT cells (1 × 105 cells/mL) were pretreated with AA (5, 10, and 20 μg/mL) for 1 h. Then, they were treated with 0.2 ng/mL IFN-γ and TNF-α for 24 h to induce inflammation. To determine the cell viability, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide assay (Promega, Madison, WI, USA) was used [15 (link)].
6
UVB Radiation Protection in HaCaT Cells
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The detailed methods have been described previously [7 (link)]. In brief, HaCaT cells were purchased from CLS Cell line service (Eppelheim, Germany) and maintained in Huons Co., Ltd. (Gyeonggi-do, Korea). We obtained the HaCaT cells from Huons, and the cells were cultured in Dulbecco's modified Eagle medium (DMEM; Gibco, Rockville, MD, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco) and 1% antibiotics (Gibco) at 37°C and 5% CO2 in a humidified incubator. The cells were plated and allowed to adhere for 24 h, and then treated with various concentrations of HU-018. After 24 h, the medium was changed and exposed to UVB radiation at a dose of 20 mJ/cm2. UVB irradiation was performed using a UVM-225D Mineralight UV display lamp (UVP, Phoenix, AZ, USA) that emitted at a wavelength of 302 nm. The strength of the UV radiation was measured using a HD2102-2 UV meter (Delta OHM, Padova, Italy).
7
Quantification of Vitamin D Receptor Gene Expression
8
Evaluating Cytotoxicity of Novel Repellent Compounds
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To test the effect of the new derivatives on cell integrity, normal
human keratinocytes (HaCaT cells, from ATCC, USA) were used to measure
cell viability after exposure to the most promising compounds, using
DEET and Icaridin as references. HaCaT cells were cultured in 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1%l -glutamine (4 mM), and 1% penicillin–streptomycin (Thermo
Fisher Scientific, Rodano, Milan, Italy). HaCaT cells were then seeded
(1 × 105 cells/well) and exposed to 10 derivatives
(6b , 9b , 12a , 13a , 15a , 16b , 16c , 17b , 17c , and 18a ) or to DEET and Icaridin,
at a final concentration of 82 μg/mL, selected to obtain a final
solvent concentration (ethanol) below 0.1%. Cytotoxicity was evaluated
after 24 h using the MTS reagent (CellTiter 96 Aqueous proliferation
assay; Promega Madison, WI, USA) as previously described.32 (link) For compounds exhibiting cytotoxicity at 24
h but of interest for their repellence, we evaluated also the HaCaT
viability after 3 and 6 h of exposure. Experiments were performed
in triplicate.
human keratinocytes (HaCaT cells, from ATCC, USA) were used to measure
cell viability after exposure to the most promising compounds, using
DEET and Icaridin as references. HaCaT cells were cultured in 5% CO2 at 37 °C in Dulbecco’s modified Eagle’s
medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 1%
Fisher Scientific, Rodano, Milan, Italy). HaCaT cells were then seeded
(1 × 105 cells/well) and exposed to 10 derivatives
(
at a final concentration of 82 μg/mL, selected to obtain a final
solvent concentration (ethanol) below 0.1%. Cytotoxicity was evaluated
after 24 h using the MTS reagent (CellTiter 96 Aqueous proliferation
assay; Promega Madison, WI, USA) as previously described.32 (link) For compounds exhibiting cytotoxicity at 24
h but of interest for their repellence, we evaluated also the HaCaT
viability after 3 and 6 h of exposure. Experiments were performed
in triplicate.
9
Cytotoxicity Evaluation of Orobol and Bentonite Composites
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The in vitro cytotoxicities of orobol, Bt-PC and Bt-PC@Orobol (2:2:1) composite formulation were evaluated in human keratinocyte (HaCaT) cells (Thermo Fisher Scientific, Massachusetts, USA). Briefly, cells were seeded on 96-well plates at 1.0 × 104 cells/100 µL in complete Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.5% penicillin‒streptomycin. After applying various concentrations of orobol solution (3 × 10−4 ~ 3 × 101 µg/mL) or bentonite composite formulation (Bt-PC or Bt-PC@Orobol) at 1 × 10−3 ~ 3 × 102 µg/mL, the cells were incubated for 24 h or 48 h at 37 °C. Then, the cytotoxicity was assessed by a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Milan, Italy) according to the manufacturer’s instructions. The absorbance was read in a microplate reader (Multiskan Microplate Spectrophotometer; Thermo Fisher Scientific, Massachusetts, USA) at 492 nm.
10
Evaluating Keratinocyte Responses to Herbal Extracts
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HaCaT cells (immortalized human keratinocytes) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). DMEM medium supplemented with 10% FBS was used for the culturing of the cells, which were maintained at 37 °C in a humidified 5% CO2 atmosphere. Cells were seeded in 96 and 48-well plates (8 × 103 and 1.5 × 104 cells per well, respectively) for the MTT, PI incorporation, and cell migration assays. The cultures were kept at 37 °C in a humidified 5% CO2 atmosphere for 24 h and were further treated with ASE (previously dried and resuspended in dimethyl sulfoxide (DMSO)) and NEASE at 0.625, 1.25, 2.5, 5, and 10 μg/mL concentrations. After 24 h of treatment, cell viability, death by necrosis, and migration were determined as follows.
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