HPK were seeded in a 96-well plate (6000 cells per well) over night and were incubated with basal Keratinocyte-SFM medium (Thermo Fisher, Darmstadt, Germany) for 24 h. Then, the cells were incubated for a further 24 h with the plant extracts before the cell viability was assessed with the CellTiter-Glo2.0 Assay (Promega, Walldorf, Germany) according to the manufacturer’s protocol. The method is based on the bioluminescent measurement of ATP that is present in metabolically active cells. Luciferase catalyzes the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration and is measured using a scanning multiwell spectrophotometer (Sirius HT from BioTek, Bad Friedrichshall, Germany).
Basal keratinocyte sfm medium
Manufactured by Thermo Fisher Scientific
Sourced in Germany, United States
Basal Keratinocyte-SFM medium is a serum-free, low-calcium medium developed for the culture and growth of human epidermal keratinocytes. It is formulated to maintain keratinocytes in a basal, undifferentiated state.
Automatically generated - may contain errors
Lab products found in correlation
Bovine pituitary extract, by Thermo Fisher Scientific (2 mentions)
Sirius ht, by Agilent Technologies (1 mentions)
Celltiter glo 2.0 assay, by Promega (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Keratinocyte sfm medium, by Thermo Fisher Scientific (1 mentions)
Recombinant human egf, by Thermo Fisher Scientific (1 mentions)
H1299, by American Type Culture Collection (1 mentions)
Human recombinant epidermal growth factor, by Thermo Fisher Scientific (1 mentions)
Stempro human adipose derived stem cells, by Thermo Fisher Scientific (1 mentions)
6 protocols using basal keratinocyte sfm medium
1
Evaluating Plant Extracts' Effects on Keratinocyte Viability
2
Isolation of Mouse Keratinocytes for IL-36α Stimulation
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8- to 12-weeks-old wildtype mice were sacrificed and tails were wiped with 70% isopropanol. In order to isolate mKC, the skin was separated from the muscle and incubated overnight at 4°C in equal amounts of basal keratinocyte-SFM medium (Cat.17005042 Gibco, Thermo Fisher Scientific) and 5 U/mL dispase I solution (Cat.07913, StemCell). The next day, the epidermis was separated from the dermis and incubated for 15 min with 0.05% trypsin-EDTA solution (Cat.25300-054, Thermo Fisher Scientific) at room temperature (RT). Stopping the reaction with DMEM medium supplemented with 10% FCS, cells were carefully washed out and transferred to a collection tube using a 100 µm cell strainer. After centrifugation (1000 rpm, 5 min), the pellet was resuspended in complete keratinocyte-SFM medium (containing BPE and EGF), supplemented with 0.05 M CaCl2. Cells were seeded on collagen type I-coated 6-well plates (Cat.A1064401, Thermo Fisher Scientific). When the plates reached a confluency of 70-80% confluency, cells were starved overnight (SFM Medium without CaCl2 and supplements) and subsequently treated with 100 ng/mL IL-36α (Cat.555902, Biolegend) for 1.5 h, in the presence of either 50 µg/mL IgG or anti-IL36R antibody.
3
Psoriasis-like Human Keratinocyte Model
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Human primary keratinocytes (HPK) were prepared from adult skin of reduction surgery (approved by the ethics committee of the University Medical Center Freiburg, Certificate No EK432/18) and cultured according to the method of Rheinwald and Green [49 (link)] in Keratinocyte-SFM medium (Thermo Fisher, Darmstadt, Germany). To generate psoriasis-like HPK, cells were first grown in basal Keratinocyte-SFM medium (Thermo Fisher, Darmstadt, Germany) and then incubated with psoriasis cytokines (IL-17A, IL-22 and TNF-α, 20 ng/mL each) for 24 h. All cells were cultured at 37 °C in a humidified atmosphere with 5% CO2. For extract treatment, the cells were incubated with the extracts (1 µg/mL HL (Humulus lupulus), 1 µg/mL HP (Hypericum perforatum), 2 µg/mL CA (Curcuma amara)) or 0.3 µg/mL dithranol for 2 h before the additional stimulation with the psoriasis cytokines was performed for 24 h.
4
Isolation and Expansion of hADSCs
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For the isolation of ADSCs from humans, hADSCs were isolated from subcutaneous adipose tissue as previously described [50 (link)]. Human adipose tissue was obtained from patients with approval from the ethical committee at the Kaohsiung Medical University Hospital (IRB number: KMUH-IRB-E(II)-20150193). After obtaining informed consent, subcutaneous adipose tissue from the gluteal area was taken from patients during plastic surgery. After 3 g of subcutaneous adipose tissue was isolated from humans, it was minced with scissors. The minced adipose tissues were digested with 1 mg/mL of type IA collagenase (125 units/mg) at 37 °C under 5% CO2 for 24 h. The digested tissue was centrifuged at 1000 rpm for 5 min, and the pellet was washed twice with PBS. The K-NAC medium is composed of Keratinocyte-SFM Basal Medium (Gibco-BRL, Rockville, MD, USA) supplemented with 25 mg bovine pituitary extract (BPE) (Gibco-BRL, Rockville, MD, USA), 2.5 µg human recombinant epidermal growth factor (rEGF) (Gibco-BRL, Rockville, MD, USA), 2 mM N-acetyl-L-cysteine, 0.2 mM L-ascorbic acid 2-phosphate sesquimagnesium salt, and 5% fetal bovine serum (FBS) [51 (link)]. The pellet was then resuspended in K-NAC medium and plated in a 100 mm culture dish. The K-NAC medium used in this study helped isolate and expand hADSCs, according to previous reports. In this study, we used hADSCs at passages 6–8 (P6–P8).
5
NSCLC and Normal Bronchial Cell Culture
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Human NSCLC lines H157, H226, H292, H358, H460, H522, H596, H1299, H1792, H1944, Calu-1, SK-MES, and A549, were purchased from the American Type Cell Culture (Rockville, MD). Cells were cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM l -glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin at 37 °C in the presence of 5% CO2. The normal human bronchial epithelial cell lines HBE1, HBE2, HBE3, HBE4 and HBE5 (kindly provided by Dr. John Minna of The University of Texas Southwestern Medical Center, Dallas, TX) were maintained in Keratinocyte-SFM basal medium (GIBCO) supplemented fresh at the time of use with 0.1–0.2 ng/ml EGF human recombinant and 20–30 μg/ml bovine pituitary extract. The cells were cultured and treated at 37 °C in a humidified incubator containing 5% CO2 and maintained free of mycoplasma as determined by Hoechst staining.
6
Expansion of Human Adipose-Derived Stem Cells
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The human ADSCs used in the present study were purchased from StemPro (StemPro Human Adipose-Derived Stem Cells, Gibco BRL, Thermo Fisher Scientific, Waltham, MA, USA). Human ADSCs were cultured in K-NAC medium for ADSC expansion per the protocol presented in our previous papers [31 (link),32 (link)]. The K-NAC medium comprised a keratinocyte SFM basal medium (Gibco BRL, Rockville, MD, USA) supplemented with 25 mg of bovine pituitary extract (Gibco BRL), 2.5 µg of human recombinant epidermal growth factor (Gibco BRL), 2-mM N-acetyl-L-cysteine, 0.2-mM L-ascorbic acid 2-phosphate sesquimagnesium salt, and 5% fetal bovine serum (FBS) [31 (link),32 (link)]. The cells were cultured and expanded in a humidified atmosphere with 5% CO2 at 37 °C until a sufficient number of cells was acquired.
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