The cytotoxicity activity of bacteria was studied using the human keratinocyte cell line HaCaT (Eppelheim, Germany) as previously described (N’Diaye et al., 2016 (link)). Briefly, HaCaT cells were grown at 37°C under 5% CO2 atmosphere, in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Levallois-Perret, France) supplemented with 10% fetal calf serum and 1% antibiotic cocktail (HyClone Thermo Scientific, Illkirch, France). Cells were used between passages 41 and 65. One day before use, HaCaT cells were starved of antibiotic and fetal calf serum. Cells were incubated for 24 h with bacteria at a multiplicity of infection (MOI) of 10:1. Bacterial cytotoxicity was determined by measurement of lactate dehydrogenase (LDH) release by HaCaT cells. LDH is a stable cytosolic enzyme that diffuses into the culture medium upon cell lysis and was measured using a Cytotox 96 assay (Promega). Results are the mean of three independent experiments each done in independent triplicate measurement.
Hacat
Manufactured by Lonza
Sourced in United States
HaCaT is a cell line derived from human keratinocytes. It is a well-established in vitro model commonly used in dermatological and skin-related research.
Automatically generated - may contain errors
Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Lonza (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Hacat, by American Type Culture Collection (3 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Dmem medium, by Lonza (2 mentions)
Normal human dermal fibroblasts (nhdf), by Lonza (2 mentions)
Cytotox 96 assay, by Promega (1 mentions)
Hacat, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
L glutamine, by Lonza (1 mentions)
Hacat, by Addexbio (1 mentions)
Ultraglutamine 1, by Lonza (1 mentions)
Human umbilical vein endothelial cells (huvec), by Lonza (1 mentions)
Rpmi 1640, by Lonza (1 mentions)
Human umbilical vein endothelial cells (huvec), by American Type Culture Collection (1 mentions)
Gentamicin sulfate, by Lonza (1 mentions)
Emem medium, by Lonza (1 mentions)
L glutamine, by Merck Group (1 mentions)
12 protocols using hacat
1
Cytotoxicity Assessment of Bacteria
2
Inducing Differentiation in HaCaT Cells
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The immortalized human KCs cell line HaCaT was purchased from AddexBio (San Diego, CA) (number T002000). HaCaT cells were maintained in DMEM with 4.5 g/l glucose with L-Glutamine (number BE12-604F, Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (number F7524, Invitrogen, Waltham, MA) and 100 units/ml of penicillin and 100 μg/ml of streptomycin (number P0781, Sigma-Aldrich, St. Louis, MO). To obtain cells exhibiting a basal KC phenotype, HaCaT cells were cultured for at least 4 weeks in calcium-free DMEM (number 21068028, Gibco, Waltham, MA) supplemented with 10% fetal bovine serum (number F7524, Invitrogen), 2 mM glutamine (number 25030081, Gibco), 100 units/ml penicillin, 100 μg/ml streptomycin, and calcium chloride to a final concentration of 0.07 mM (low calcium medium). Cells were induced to differentiate by adding calcium chloride to the culture medium to a final concentration of 1.8 mM (HC-containing medium). The medium was changed every 3 days for different time periods (3, 6, or 9 days).
3
Culturing Human Endothelial and Keratinocyte Cells
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Human umbilical vein endothelial cells (HUVEC) and human keratinocytes (HaCaT) were purchased from American Type Culture Collection (Manassas, VA). HUVEC and HaCaT were maintained in DMEM and U937 cells were cultivated in RPMI 1640, (Lonza). Media were supplemented with 10% fetal calf serum (FCS) (PAA Laboratories, Austria), 1% ultraglutamine 1 (Lonza), and 0.055% gentamicin sulfate (Lonza). Human cells were incubated at 37°C with 5% CO2 and passaged every three days. Cells with less than 30 passages were used in the experiments.
4
Larynx Cancer Cell Line Protein Expression
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Protein expression studies (Western blot and immunofluorescence [IF]) were conducted using the reference Larynx Epidermoid Carcinoma 2 (HEp-2) (collection of cell lines of the Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland) adherent laryngeal cancer cell line, and the Normal Human Keratinocyte cell line (HaCaT) (The American Type Culture Collection, Manassas, VA, USA), an adult normal immortalized human keratinocyte cell line, was used as the control. HEp-2 cells were cultured in EMEM medium (Lonza, Basel, Switzerland). HaCaT cells were cultured in DMEM medium (Lonza). Both media were supplemented with 10% fetal bovine serum (FBS) (Merck, Darmstadt, Germany), 1% penicillin/streptomycin (Merck), and L-glutamine (Merck). The HERA cell incubator (Heraeus, Hanau, Germany) was used to maintain constant conditions of cell cultures, i.e., a temperature of 37 °C, 5% CO2 concentration, and a 95% humidity level.
5
Cell Culture of Melanoma and Keratinocyte Lines
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The human melanoma cells lines A375 were bought from Sigma–Aldrich (Milan, Italy). WM1862, WM983A and WM983B cell lines were purchased from Rockland (Limerick, Ireland). Human keratinocytes (HaCaT) were purchased from Lonza (Basel, Switzerland)). All cell lines were cultured in RPMI 1640 medium with GlutaMAXTM and were supplemented with 10% heat-inactivated fetal calf serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 10 mM HEPES buffer (all from Gibco; New York, NY, USA). Cells were grown at 37 °C in a humidified incubator under 5% CO2. All cell lines used in this study were characterized by the cell bank where they were purchased. ERU was purchased from Cayman Chemicals (Michigan, CA, USA).
6
Stable Transfection of HaCaT Cells with CRBPI Gene
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Immortalized human keratinocyte cells HaCaT, (Lonza, Milano, Italy) maintained in D-MEM (Lonza Bio Pharma AG, Switzerland) were transfected by using a vector pTargeT Mammalian expression system carrying the whole sequence of CRBPI gene (NM_002899) and the gene for G418 resistance (Promega, Italy), or the G418-resistance gene alone. After 20 days, stable transfected clones were collected in G418-containing medium and tested by PCR and western blot. The correct plasmid sequence was confirmed by Sanger sequencing. Experimental procedures were repeated by using different transfected clones, which gave similar results.
7
Culturing HaCaT and NHDF Cells
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The human keratinocyte cell line, HaCaT, was purchased from CLS (#300493, Cell Lines Service, Eppelheim, Germany). Normal human dermal fibroblasts, neonatal (NHDF), were purchased from Thermo Fisher Scientific (#C-004-5C, Waltham, MA, USA). HaCaT cells and NHDF were cultured in Dulbecco’s modified Eagle’s medium (DMEM; #12-604F, Lonza, Walkersville, MD, USA) containing 10% fetal bovine serum (#10082-147, Thermo Fisher Scientific), 100 U/mL potassium penicillin and 100 mg/mL streptomycin sulfate (#17-602E, Lonza) at 37 °C in a humidified 5% CO2 incubator. The rate of cell viability was quantified using the Cell Counting Kit-8 (CCK-8) reagent (#CK04-11, DOJINDO, Tokyo, Japan) according to the manufacturer’s instructions.
8
Cytotoxicity Assay of PECS on Cell Lines
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The six cell lines, A375, H460, HT29, MCF7, HepG2, and HaCaT (Lonza, Verviers, Belgium), were kept in culture and expanded at 37 °C in a humidified atmosphere of 5% CO2 in DMEM (Dulbecco’s Modified Eagle’s Medium, Lonza, Basel, Switzerland) culture medium for HaCaT, HepG2, HT29, MCF7, and A375, and RPMI-1640 for H460, supplemented with 10% FBS, Penicillin/Streptomycin 100× (Euroclone, Devon, UK), Glutamax 100× (Invitrogen, Carlsbad, CA, USA) and non-essential amino acids 100× (Invitrogen). Phosphate buffer (PBS, phosphate-buffered saline, Ca2+ and Mg2+ free) and trypsin (Ca2+ and Mg2+ free) were supplied by Euroclone. The cells were plated 15 × 103 perwell in 96-well tissue culture plates and allowed to attach for 24 h. Then, cells were treated with PECS dissolved in culture medium supplemented with 1% FBS at different concentrations for 48 h (50 μg/mL, 80 μg/mL, 110 μg/mL, 140 μg/mL, 170 μg/mL, and 200 μg/mL). PECS were dissolved in sterile H2O at 100 mg/mL.
9
Cell culture protocol for common cell lines
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The mouse normal fibroblasts cell line Balb/3T3 (American Type Culture Collection ATCC®, Old Town Manassas, VA, USA), the normal human dermal fibroblasts line (NHDF) (Lonza, Basel, Switzerland), the human epidermal keratinocyte line (HaCaT) (DKFZ, Heidelberg, Germany) [48 (link)] and the human neuroblastoma cell line (SH-SY5Y) (ATCC®) were used in the study. The Balb/3T3 and HaCaT lines were cultured with DMEM medium (Lonza) with 10% foetal bovine serum (FBS) and 1% L-glutamine with penicillin and streptomycin solution (Sigma-Aldrich®, St. Louis, MO, USA). NHDF was grown in FGMTM Fibroblast Growth Medium BulletKitTM (Lonza). SH-SY5Y was cultured with F-12 medium (Lonza) supplemented with 10% FBS (Sigma-Aldrich®) and 1% L-glutamine with penicillin and streptomycin solution (Sigma-Aldrich®). Cell culture was maintained in 5% CO2 at 37 °C and 95% humidity. The cells were assessed twice a week, and the fresh medium was changed or passaged if confluence was approximately 70%.
10
Culturing Primary and Immortalized Keratinocytes
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Primary human epidermal keratinocytes (NHEK-Ad) and an immortal human keratinocyte cell line (HaCaT) were obtained from Lonza and the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in KBMTM-2 medium (Lonza; Basel, Switzerland) with KGMTM-2 growth supplement (Lonza), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco; Grand Island, NY, USA) at 37 °C in a humidified chamber supplemented with 5% CO2.
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