HAECs and HASMCs were purchased from PromoCell (UK) and maintained at 37°C in a humidified incubator supplemented with 5% CO2. Cells were cultured in endothelial cell growth media and smooth muscle cell growth media, respectively, containing 1% penicillin–streptomycin (Sigma‐Aldrich, UK) and supplemental mix (PromoCell, UK). In all experiments, cells were used between passages 3 and 5. After experimental treatments, cell media was collected and cells were washed once with phosphate‐buffered saline (PBS; pH 7.4; Gibco™). Radioimmunoprecipitation buffer (Sigma‐Aldrich) with protease and phosphatase inhibitors (A32959; Thermo Fisher Scientific) was added to lyse the cells and the plates were shaken at 4°C for an hour. The cells were then collected and centrifuged at 14,000g for 5 min at 4°C and cell supernatants were frozen at −80°C.
Supplemental mix
Manufactured by PromoCell
Sourced in United Kingdom, Germany
Supplemental mix is a laboratory reagent designed to supplement cell culture media. It provides additional nutrients and growth factors to support the growth and maintenance of various cell types in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Radioimmunoprecipitation buffer, by Merck Group (1 mentions)
Hasmcs, by PromoCell (1 mentions)
A32959, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
Collagen, by Advanced BioMatrix (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
Ebm medium, by Lonza (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Hydrocortisone, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
8 protocols using supplemental mix
1
Cultivation and Lysis of HAECs and HASMCs
2
Culturing Primary Human Lens Epithelial Cells
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Primary male human LECs (Hirakawa et al., 2003 (link)) were cultured under standard culture conditions (37°C and 5% CO2) on collagen (Advanced BioMatrix) coated dishes (50 μg/mL) in EBM medium (Lonza) containing 20% FBS (GIBCO), 1% penicillin/streptomycin (GIBCO), 2 mmol/L L-glutamine (GIBCO), 25 μmol/mL cAMP (Sigma-Aldrich) and 10 μg/mL hydrocortisone (Sigma-Aldrich).
Human bronchial smooth muscle cells were purchased from ScienCell and cultured under standard conditions in Smooth Muscle Cell Medium 2 containing the Supplemental Mix (both PromoCell). MeWo cells (Sigma Aldrich) were cultured under standard culture conditions in DMEM containing pyruvate (GIBCO) and 10% FBS.
Human bronchial smooth muscle cells were purchased from ScienCell and cultured under standard conditions in Smooth Muscle Cell Medium 2 containing the Supplemental Mix (both PromoCell). MeWo cells (Sigma Aldrich) were cultured under standard culture conditions in DMEM containing pyruvate (GIBCO) and 10% FBS.
3
Culturing Primary Human Cardiac Myocytes
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Primary human cardiac myocytes (PHCM) were obtained from and cultured following the manufacturer’s recommendations (PromoCell, Heidelberg, Germany). Briefly, the PHCM were cultured in myocyte basal growth medium supplemented with the supplemental mix (PromoCell, Heidelberg, Germany) containing fetal calf serum (0.05 mL/mL), recombinant human epidermal growth factor (0.5 ng/mL), recombinant human basic fibroblast growth factor (2 ng/mL) and recombinant human insulin (5 ug/mL). The cells were cultured in T75 flasks at 37 °C in the presence of 5% CO2 to approximately 80% confluency (approximately 4 × 106 cells) prior to being used in our assays.
4
Culturing Primary Human Cardiac Fibroblasts
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PHCF were obtained from and cultured following the manufacturer’s recommendations (PromoCell, Heidelberg, Germany). Briefly, the PHCF were cultured in fibroblast basal growth medium supplemented with the supplemental mix (PromoCell, Heidelberg, Germany) containing fetal calf serum (0.05 mL/mL), recombinant human epidermal growth factor (0.5 ng/mL), recombinant human basic fibroblast growth factor (2 ng/mL) and recombinant human insulin (5 ug/mL). The cells were cultured in T75 flasks at 37°C in the presence of 5% CO2 to approximately 80% confluency (approximately 4 × 106 cells) prior to being used in our assays.
5
Cell Culture Protocols for Liver and Endothelial Cells
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LX2 cells were maintained in Iscove’s Modified DMEM supplemented with 2 mM/L-glutamine, 0.1 mM/L non-essential amino acids, 1.0 mM/L sodium pyruvate and 20% foetal bovine serum (FBS). HepG2 (ATCC® HTB-52™, VA, USA) were maintained in Eagle’s Minimum Essential medium (EMEM), supplemented with Glutamax, 0.1 mM/L non-essential amino acids, 1.0 mM/L sodium pyruvate and 10% FBS. Human umbilical vein endothelial cells (HUVEC pooled; PromoCell) were cultured in low-serum (2% V/V) ECGM with a supplemental mix (PromoCell) and used between passage 3 and 5. All cells were sub-cultured every 3 days at a split ration of 1:3.
Primary Human HSCs (hHSC) were isolated from wedge sections of liver tissue, obtained from patients undergoing surgery at the Royal Free Hospital after giving informed consent (EC01.14-RF). Cells were isolated according to Mederacke et al.48 (link), with modifications for human liver49 (link).
The obtained HSCs were cultured in IMDM supplemented with 20% FBS, glutamine, nonessential amino acids 1X, 1.0 mM sodium pyruvate, 1X antibiotic-antimycotic (all Life Technologies), hereafter referred to as complete HSC medium. Experiments described in this study were performed on hHSC of at least three independent cell preparations, between passage 3 and 8.
All cells were maintained under standard conditions at 37 °C in a humidified incubator containing 5% CO2.
Primary Human HSCs (hHSC) were isolated from wedge sections of liver tissue, obtained from patients undergoing surgery at the Royal Free Hospital after giving informed consent (EC01.14-RF). Cells were isolated according to Mederacke et al.48 (link), with modifications for human liver49 (link).
The obtained HSCs were cultured in IMDM supplemented with 20% FBS, glutamine, nonessential amino acids 1X, 1.0 mM sodium pyruvate, 1X antibiotic-antimycotic (all Life Technologies), hereafter referred to as complete HSC medium. Experiments described in this study were performed on hHSC of at least three independent cell preparations, between passage 3 and 8.
All cells were maintained under standard conditions at 37 °C in a humidified incubator containing 5% CO2.
6
KCs Response to S. aureus and IFN-γ
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To determine the effect S. aureus and IFN-γ exposure on human KCs, we cultured the primary human KCs obtained from the Skin Biology and Diseases Resource Center, Penn Dermatology in KC Media 2 supplemented with 0.06 mM CaCl2 and SupplementalMix (C-20011; Promo Cell). Cells were seeded at 105 per well in 6-well plates. At 75% confluency, cells were fed with fresh media and exposed to 106S. aureus in 2 ml total media. After 18 h, cells were stimulated with 50 ng/ml IFN-γ for 48 h. Cells were detached using Cell Detachment Kit (C-41000; Promo Cell) to stain with propidium iodide and flow cytometry analysis.
7
PHCF Culture and Growth Protocol
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PHCF low passage, was grown according to the manufacturer's recommendations (PromoCell, Heidelberg, Germany). Briefly, PHCFs were cultured in fibroblast basal growth medium supplemented with the supplemental mix (PromoCell) containing fetal calf serum (0.1 ml/ml), recombinant human epidermal growth factor (0.5 ng/ml), recombinant human basic fibroblast growth factor (2 ng/ml) and recombinant human insulin (5 ug/ml). The cells were grown in T75 flasks at 37°C in the presence of 5% CO2 to a confluence of about 80% (about 4×106 cells) before being used in the T. cruzi infection and control assays.
8
Culturing Primary Human Cardiac Myocytes
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Low passage culture of primary human cardiac myocytes (PHCM) was grown according to the manufacturer’s recommendations (PromoCell, Heidelberg, Germany). Briefly, the PHCMs were cultured in myocytes basal growth medium supplemented with the supplemental mix (PromoCell, Heidelberg, Germany) containing fetal calf serum (0.05ml/ml), recombinant human epidermal growth factor (0.5ng/ml), recombinant human basic fibroblast growth factor (2ng/ml) and recombinant human insulin (5ug/ml). The cells were grown in T75 flasks at 37°C in the presence of 5% CO2 to a confluence of about 80% (about 4X106 cells) before used in the T. cruzi infection and control assays.
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