For cytotoxicity tests, normal human dermal fibroblasts (NHDF) cells (from PromoCell) at a passage number lower than 10 were grown in T25 culture flasks with 10 mL of DMEM:F12 medium (from Lonza) supplemented with 10% fetal bovine serum (FBS, Gibco), 1 mM sodium pyruvate (Lonza) and 1 % Penicillin-Streptomycin-Amphotericin B mixture (10K/10K/25 µg in 100 mL, Lonza). Cell growing was done at 37 °C and 5 % CO 2 under humidified atmosphere and medium was changed with fresh one every 4 days. NHDF cells were harvested by trypsinization with Trypsin-Versene (EDTA) mixture (Lonza), washed with phosphate buffered saline (PBS, Invitrogen) centrifuged at 200 rpm for 5 min, counted and seeded at a density of 5x10 3 cells/well in 96 well plates with 100 µL/well of the above specified medium. Cells were incubated and were allowed to adhere in humidified atmosphere at 37°C until the next day.
Penicillin streptomycin amphotericin b mixture
Manufactured by Lonza
Sourced in United States, Switzerland, Germany
Penicillin-Streptomycin-Amphotericin B mixture is a broad-spectrum antibiotic solution used in cell culture applications. It contains a combination of penicillin, streptomycin, and amphotericin B, which collectively inhibit the growth of bacteria, fungi, and other microorganisms in cell culture media.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (3 mentions)
L glutamine, by Lonza (3 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
Normal human dermal fibroblasts (nhdf), by PromoCell (2 mentions)
Alpha mem medium, by Lonza (2 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (2 mentions)
Trypsin versene edta mixture, by Lonza (1 mentions)
Nhdf cells, by PromoCell (1 mentions)
Sodium pyruvate, by Lonza (1 mentions)
Dmem f12 medium, by Lonza (1 mentions)
Fluostar omega microplate reader, by BMG Labtech (1 mentions)
Hepatocyte growth factor (hgf), by CLS Cell Lines Service (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
Dialysis tubing cellulose membrane, by Merck Group (1 mentions)
Dmem high glucose, by Lonza (1 mentions)
Dulbecco s modified eagle s medium dmem, by Lonza (1 mentions)
15 protocols using penicillin streptomycin amphotericin b mixture
1
Cytotoxicity Assay with NHDF Cells
2
Biocompatibility Assessment of Extracts
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Human gingival fibroblasts (HGF, CLS Cell Lines Service GmbH, Eppelheim, Germany) were seeded into 96-well tissue, culture-treated plates at a density of 0.5 × 105 cells/mL and allowed to adhere overnight in complete cell culture medium: MEM α medium with 10% fetal bovine serum (FBS, both from Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% Penicillin-Streptomycin-Amphotericin B mixture (10 K/10 K/25 μg, Lonza, Basel, Switzerland). Extracts were done for every investigated sample in complete cell culture medium at 10 mg/mL, for 24 h, at 37 °C. The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS, Promega, Madison, WI, USA) was used to assess the biocompatibility of samples’ extracts, according to the manufacturer instructions and ISO 10993-5:2009(E). Cells were incubated for 24 h with fresh, complete medium (Control) or different concentrations of samples’ extracts (2.5 mg/mL, 5 mg/mL, 7.5 mg/mL, and 10 mg/mL). After incubation with MTS reagent (3 h), absorbance readings were done at 490 nm on a FLUOstar® Omega microplate reader (BMG LABTECH, Ortenberg, Germany). Experiments were done in triplicate, and treated cell viability was expressed as a percentage of the Control cells’ viability (means ± standard deviation). Data were statistically analyzed by the independent two-tailed (Student’s) t-test, considering p < 0.05 to be statistically significant.
3
Culturing Human Dermal Fibroblasts
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Human dermal fibroblasts, adult (HDFa) was obtain from Gibco (Gibco, Life Technologies Corp. C-013-5C, Carlsbad, CA). HDFa were cultured with DMEM (Sigma-Aldrich, Saint-Louis, MO, USA) supplemented with 10% heat-inactivated FBS (Sigma-Aldrich, Saint-Louis, MO, USA), 2% L-glutamine (Lonza, Basel, Switzerland) and 1% Penicillin-Streptomycin-Amphotericin B Mixture (Lonza, Basel, Switzerland). For the experiments, HDFa were used from passage P3 to passage P7. HDFa were seeded at a density of 5000 cells/cm² and maintained at 37 °C in 5% CO2. The medium was changed twice a week. Cell confluence at the time of experiment was approximately 80%.
4
Quantifying M. tuberculosis-specific CD4+ T cell Responses
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On Day 0, isolated CD4+ T cells and CD8- PBMCs were mixed 2:1 (8-12×106) at 4×106/ml. Each M. tuberculosis antigen peptide pools 0.1ug/ml were added to RPMI 1640 supplemented with 2 mM L-glutamine, 100 U/ml Penicillin-Streptomycin-Amphotericin B Mixture (Lonza), 10% fetal bovine serum (Gibco), 500U/ml IFN-γ (LG Chemical) and 200U/ml hIL-2 (JW CreaGene) and incubated in 5% CO2, 37°C. On Day 1, 4, and 7, hIL-2 was added to medium 200U/ml. On Day 11, cells were sub-cultured to replenish the medium at 2×106/ml with hIL-2 200U/ml. On Day 14, cells were harvested and measured IFN-γ secretion by ELISPOT assay. The 5×104 cultured CD4+ T cells and 5×103 antigen-pulsed or not aAPC were co-incubated for 20h at 37°C. M. tuberculosis antigens-specific responses of cultured CD4+ T cell responses were measured by IFN-γ ELISPOT assay as described in cultured ELISPOT assay. To compare ex vivo and cultured ELISPT, the frequencies of each ELISPOT were adjusted to account for differences in cell numbers and proliferation folds among the donors. It was calculated as (SFCs/5×104 CD4+ T cells) × (1×106) × proliferation fold. These were determined as adjusted Spots-Forming Cells (aSFCs) by cultured ELISPOT.
5
Culturing HaCaT Keratinocytes for Experimentation
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HaCaT was obtained from CLS (Cell Line Service, Eppelheim, Germany, ref. 3300493). HaCaT was cultured with DMEM (Sigma-Aldrich, Saint-Louis, MO, USA) supplemented with 10% heat-inactivated FBS (fetal bovine serum) (Sigma-Aldrich, Saint-Louis, MO, USA), 2% L-glutamine (Lonza, Basel, Switzerland), and 1% Penicillin-Streptomycin-Amphotericin B Mixture (Lonza, Basel, Switzerland). HaCaT cells were seeded at a density of 10 000 cells/cm2 and maintained at 37 °C in 5% CO2. The medium was changed twice a week. The cell confluence at the time of experiment was approximately 80%.
6
Chitosan-Hyaluronic Acid Bioactive Dressings
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Chitosan with a medium molecular weight (200–300 kDa, DDA > 85%, viscosity of 200–800 cP) and hyaluronic acid sodium salt from Streptococcus equi, with low molecular weight (100–230 kDa) were pharmaceutical grade; lactic acid (99%), sodium periodate (99.8%), ethylenglicol anhydrous (98%) were analytical grade; allantoin (Ala), fusidic acid (FA) and coenzyme Q10 (CoQ10) were micronized pharmaceutical grade powders. All these chemicals were purchased from Sigma-Aldrich (Merck Group, Schnelldorf, Germany) and were used without any further purification. Dialysis tubing cellulose membrane with a molecular weight cut off of 14,000 Da was also purchased from Sigma-Aldrich. Human fibroblasts (HGF, CLS Cell Lines Service GmbH, Eppelheim, Germany), MEMα medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 1% Penicillin-Streptomycin-Amphotericin B mixture (Lonza, Basel, Switzerland) were also used. Gram-negative (Escherichia coliATCC 25922) and Gram-positive (Staphylococcus aureusATCC 25923) bacterial strains aswell as pathogenic yeast (Candida albicansATCC 90028) were provided by Mecconti, Poland.
7
Isolation and Culture of Breast Milk Cells
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Breast milk was diluted 1:2 with Dulbecco's modified Eagle's medium (DMEM) (Lonza Bioproducts, Verviers, Belgium) and centrifuged at 400g for 10 min. The cell pellet was washed twice with phosphate-buffered saline (PBS) (Lonza Bioproducts). Final cell pellet was resuspended in complete culture medium: high glucose DMEM (4.5 g/L glucose with L-gluta- mine, Lonza Bioproducts) with 10% fetal bovine serum (FBS) and 1% Penicillin Streptomycin Amphotericin B mixture (10 IU/10 IU/25 mg, Lonza Bioproducts). Cells were cultured at a concentration of 5 3 10 6 per 25-cm 2 culture flask and incubated at 37 8C in 5% humidified CO 2 in a CO 2 incubator (Heraeus, Hanau, Germany). The media were changed every 48 h for 12-14 days as primary culture. At 80% confluence, cells were passage using Trypsin/ethylenediaminetetraacetic acid (EDTA). Forth-passage (p4) cells were used for experiments (8) .
8
Antioxidant Activity of Iron-Polyphenol Complexes
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Chemical reagents were of analytical grade and used as received without further purification. Cerium(III) nitrate hexahydrate (Ce(NO3)3∙6H2O), ferric chloride (FeCl3∙6H2O), ferrous chloride (FeCl2∙4H2O), branched polyethylenimine of 1.8 kDa, 25% glutaraldehyde aqueous solution (GA), 25% ammonium solution and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich; normal human dermal fibroblasts (NHDF) from PromoCell; CellTiter 96® AQueous One Solution Reagent, containing tetrazolium compound [3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)] and electron coupling reagent (phenazine ethosulfate; PES) from Promega; alpha-MEM medium and 1% Penicillin-Streptomycin-Amphotericin B mixture from Lonza; 10% fetal bovine serum (FBS) from Gibco; T75 cell culture flasks from Corning; Antioxidant Assay Kit CS0790 from Sigma-Aldrich (St. Louis, MO, USA).
9
Bovine in vitro cytokine response
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A total of 500 mL of heparinized blood samples was cultured in triplicate within two hours of collection in 24-well plates (Thermo Fisher Scientific, USA) with 500 mL RPMI supplemented with 10% foetal bovine serum (Thermo Fisher Scientific, Waltham, USA) and 1% penicillin-streptomycin-amphotericin B mixture (Lonza, Belgium) referred to as complete medium (CM), as described previously (Vázquez et al., 2019 (link)). Each blood replicate was also supplemented with N. caninum soluble extract antigen obtained from Nc-Spain7 tachyzoites (Alvarez-García et al., 2003 (link)) at 5 μg/mL, concanavalin (ConA) (Sigma-Aldrich, Spain) at 5 μg/mL as a positive control, and PBS as negative control. After 24 h of incubation (37°C, 5% CO2), culture supernatants were collected by centrifugation of plates at 1000 × g for 10 min at 4°C and stored at −80 °C until laboratory analysis for the evaluation of IFN-γ and IL-4 release.
Cytokine release was measured with commercial bovine IFN-γ and bovine IL-4 ELISA kits (Mabtech AB, Nacka Strand, Sweden) following the manufacturer’s guidelines. The cytokine concentrations were calculated by interpolation from a standard curve produced with recombinant cytokines provided with the kits. The colour reaction was developed by the addition of 3,3’,5,5’-tetramethylbenzidine substrate (TMB, Sigma-Aldrich, Spain). Plates were read at 450 nm.
Cytokine release was measured with commercial bovine IFN-γ and bovine IL-4 ELISA kits (Mabtech AB, Nacka Strand, Sweden) following the manufacturer’s guidelines. The cytokine concentrations were calculated by interpolation from a standard curve produced with recombinant cytokines provided with the kits. The colour reaction was developed by the addition of 3,3’,5,5’-tetramethylbenzidine substrate (TMB, Sigma-Aldrich, Spain). Plates were read at 450 nm.
10
Simian Rotavirus Infection in MA104 Cells
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The African Green Monkey kidney (MA104) cell line was maintained in Dulbecco’s modified Eagle medium (DMEM; Gibco), supplemented with 5% (v/v) foetal bovine serum (FBS; Gibco), 1% (v/v) Penicillin-Streptomycin-Amphotericin B Mixture (10,000 U, 10,000 μg and 25 μg/ml; Lonza) and 1% (v/v) nonessential amino acids (NEAA; Lonza) at 37°C and 5% CO2. Subconfluent cells were supplemented with 50 μM GLA (Thermo Fischer Scientific) for 24 h according to Tanaka et al. (2001) (link). Rotavirus simian agent 11 strain (SA11; Mlera et al., 2013 (link)) was used to infect MA104 cells and viral yield was determined using 50% tissue culture infectious doses (TCID50; Reed and Muench, 1938 (link)). To facilitate infection, pancreatic porcine trypsin type IX (1 μg/ml; Sigma-Aldrich) was added during all viral replication experiments.
The non-specific cyclooxygenases inhibitor, indomethacin; the COX-1-specific inhibitor, SC-560; the COX-2-specific inhibitor, Celecoxib; cytoplasmic phospholipase A2 inhibitor, CAY10502 and PGE2 were obtained from Sigma-Aldrich and resuspended in 100% (v/v) dimethyl sulfoxide (DMSO; Sigma-Aldrich). Cellular toxicity for the inhibitors and DMSO was evaluated with the XTT assay (Sigma-Aldrich;Supplementary Figure S1 ). Final DMSO concentration did not exceed 1%.
The non-specific cyclooxygenases inhibitor, indomethacin; the COX-1-specific inhibitor, SC-560; the COX-2-specific inhibitor, Celecoxib; cytoplasmic phospholipase A2 inhibitor, CAY10502 and PGE2 were obtained from Sigma-Aldrich and resuspended in 100% (v/v) dimethyl sulfoxide (DMSO; Sigma-Aldrich). Cellular toxicity for the inhibitors and DMSO was evaluated with the XTT assay (Sigma-Aldrich;
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