Male Sprague Dawley rats were killed by cervical dislocation in accordance with schedule 1 of the U.K. Animals (Scientific Procedures) Act 1986 and Directive 2010/63/EU of the European Parliament and with the approval of the University of Bristol. Methods used to culture VSMCs and ECs are described in detail in the Supplement. Human aortic VSMCs (HuVSMCs) and human umbilical endothelial cells (HUVC) at passage 2–8 were purchased from Promocell. All experiments were replicated for the number of time shown in the text and figures using different batches of cells that were prepared from different animals/donors. Cultures of rat aortic VSMCs (RaVSMCs) were prepared as previously described [22 (link)] and cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal bovine serum, 100 U/ml penicillin/streptomycin and 2.5 mM l -glutamine. Human VSMC were cultured in human smooth muscle cell growth medium 2 (Promocell), supplemented with the supplement pack (Promocell), 100 U/ml penicillin/streptomycin and 2.5 mM l -glutamine. HUVEC were cultured in Endothelial Cell Growth Medium 2 supplemented with the supplied supplement pack (Promocell), 100 U/ml penicillin/streptomycin and 2.5 mM l -glutamine.
Supplementpack
Manufactured by PromoCell
Sourced in Germany
The SupplementPack is a laboratory equipment product designed to facilitate the handling and organization of various supplements. It provides a convenient storage and dispensing solution for powdered or liquid supplements used in research and development activities.
Automatically generated - may contain errors
Lab products found in correlation
Huvecs, by PromoCell (2 mentions)
Minimum essential medium (mem), by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Ebm mv2, by PromoCell (1 mentions)
C 12217, by PromoCell (1 mentions)
Supplementmix, by PromoCell (1 mentions)
Normal hbecs, by Lonza (1 mentions)
Imr 90, by American Type Culture Collection (1 mentions)
Airway epithelial cell growth medium, by PromoCell (1 mentions)
Gelatin from porcine skin, by Merck Group (1 mentions)
Ebm 2, by PromoCell (1 mentions)
Human smooth muscle cell growth medium 2, by PromoCell (1 mentions)
Penicillin streptomycin, by PromoCell (1 mentions)
Endothelial cell growth medium 2, by PromoCell (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Endothelial cell basal medium 2, by PromoCell (1 mentions)
Egm 2 singlequot, by Lonza (1 mentions)
Cho k1, by Thermo Fisher Scientific (1 mentions)
Cho k1, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
13 protocols using supplementpack
1
Isolation and Culture of Vascular Cells
2
Culturing Human Dermal Lymphatic Endothelial Cells
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Human dermal lymphatic endothelial cells (LECs) from three different donors (n = 3) were purchased from PromoCell (C-12217, PromoCell GmbH, Heidelberg, Germany). Cryopreserved cells were thawed and expanded in an expansion medium, which consists of Endothelial Cell Basal Medium MV2 (EBM MV2, PromoCell GmbH, Heidelberg, Germany) supplemented with SupplementMix (C-39226, PromoCell, Heidelberg, Germany), containing a company-stated optimal formula of 5% fetal calf serum (FCS), 5 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL insulin-like growth factor (IGF), 0.5 ng/mL vascular endothelial growth factor 165 (VEGF-A), 1 μg/mL ascorbic acid, and 0.2 μg/mL hydrocortisone, and were incubated at 37 °C and 5% CO2. Ensuing experiments were all carried out between the 3rd–5th cellular passages. The negative control (=basal) media consisted of Endothelial Cell Basal Medium MV2 (EBM MV2, PromoCell GmbH, Heidelberg, Germany) supplemented with 1 μg/mL ascorbic acid, 0.2 μg/mL hydrocortisone, and 1% FCS from the SupplementPack (C-39221, PromoCell, Heidelberg, Germany). For the positive control, we used the expansion media tested and proven by the company to be optimal for LECs. HPS and NS were used as undiluted (100%) and diluted with basal media at 0.1%, 1%, 10%, and 40% final concentrations for the following experiments. PRP was not diluted.
3
Culturing Human Lung Fibroblasts and Epithelial Cells
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Human fetal lung fibroblasts (IMR-90) were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in minimum essential medium (MEM) (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum (FBS; MP Biomedicals, Santa Ana, CA, USA). Normal HBECs (Lonza, Basel, Switzerland) were grown in Airway Epithelial Cell Growth Medium with SupplementPack (PromoCell, Heidelberg, Germany).
4
Multifunctional Biomaterial Hydrogel for Tissue Engineering
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PCL (Mn average 80,000), Pluronic® F-127 (PL), Sodium Alginate (AG) and gelatin from porcine skin (GL) were purchased from Sigma-Aldrich. 50:50 PLGA (Inherent Viscosity = 0.55–075 dL/g) was purchased from DURECT corporation (Cupertino, US). Gelatin crosslinking agent γ-glycidoxypropyl-trimethoxysilane (GPTMS) from Sigma-Aldrich. Endothelial basal medium-2 (EBM-2, cat# C-22211, Promocell) was used for in vitro specific assays, while complete EGM-2, consisting of EBM-2 supplemented with SupplementPack (cat#: C-39211, PromoCell), was used to culture APCs and HUVECs. Fetal bovine serum (FBS) was obtained from Hyclone (UT, USA). Phosphate buffer saline (PBS), penicillin and streptomycin were purchased from Gibco BRL, Invitrogen Corp., (Carlsbad, CA, USA).
5
Propagation and Purification of Human RSV
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Human small airway epithelial cells (hSAEC) (38 (link)) were cultured in Small Airway Epithelial Cell Basal Medium (C-21270, Promo Cell), supplemented with Supplement Pack (C-39170, Promo Cell). HEp-2 cells (CCL-23, ATCC) were grown in Minimum Essential Medium (MEM) (Gibco) containing 10% fetal bovine serum (Life Technologies), 100 units/ml penicillin (Gibco), and 100 μg/ml streptomycin (Gibco).
The human RSV A2 strain (VR-1544, ATCC) was propagated in HEp-2 cells at 80% confluence, washed with PBS without Ca2+ and Mg2+ and the virus was added at a multiplicity of infection of 0.1 plaque forming units (PFU)/cell diluted in serum free medium for 1 h at 37 °C. Complete medium was then added to the culture for prolonged inoculation at 37 °C. When high cytopathic effects were observed (days 4–7 p.i.), cells were scraped into the medium, cell debris was separated, and virus was purified on discontinuous sucrose gradients as described previously (39 , 40 ). Aliquots of sucrose-purified (cytokine and lipopolysaccharide-free) RSV virion suspensions were stored at −80 °C until used. Virus titers were determined via plaque assay as previously described (41 (link)).
The human RSV A2 strain (VR-1544, ATCC) was propagated in HEp-2 cells at 80% confluence, washed with PBS without Ca2+ and Mg2+ and the virus was added at a multiplicity of infection of 0.1 plaque forming units (PFU)/cell diluted in serum free medium for 1 h at 37 °C. Complete medium was then added to the culture for prolonged inoculation at 37 °C. When high cytopathic effects were observed (days 4–7 p.i.), cells were scraped into the medium, cell debris was separated, and virus was purified on discontinuous sucrose gradients as described previously (39 , 40 ). Aliquots of sucrose-purified (cytokine and lipopolysaccharide-free) RSV virion suspensions were stored at −80 °C until used. Virus titers were determined via plaque assay as previously described (41 (link)).
6
Isolation and Culture of HUVEC Cells
7
Cell Culture Protocols for Various Cell Lines
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HUVECs were purchased from PromoCell. The cells were maintained in EGM-2 basal medium with SupplementPack (complete EGM, PromoCell) at 37 °C in a humidified atmosphere and 5% CO2. Human cervical cancer HeLa 229, human lung fibroblast MRC-5, and chinse hamster ovary CHO-K1 cells were obtained from Japanese Collection of Research Bioresources cell bank. The cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS, Biowest) (for HeLa 229 and MRC-5; Thermo Scientific) or Ham’s F-12 medium/10% FBS (for CHO-K1; Thermo Scientific) at 37 °C in a humidified atmosphere and 5% CO2.
8
Culturing HaCaT and NHEK Cells with EGCG
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HaCaT cells, a naturally immortalized keratinocyte cell line derived from primary human epithelium, were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium/high glucose (Gibco, Carlsbad, CA, USA) containing 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco) in a humidified atmosphere containing 5% CO2 at 37 °C. Normal human epidermal keratinocytes (NHEK) from neonatal foreskin (Promocell, Heidelberg, Germany) were cultured in keratinocyte growth medium 2 with a supplement pack (Promocell) and 1% penicillin/streptomycin (Gibco). Epigallocatechin-3-gallate (EGCG) was purchased from Sigma-Aldrich (St. Louis, MO, USA). EGCG stock solution was prepared in sterile distilled water at 10 and 20 mM.
9
Modeling Psoriasis In Vitro
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HEK was isolated from circumcised foreskins of adolescent volunteers as described previously [45 (link), 46 (link)] and cultured in Keratinocyte Growth Medium 2 supplemented SupplementPack (Promo Cell, Germany). The HaCaT cells and HUVECs were purchased from Procell (Wuhan, China) and cultured in Roswell Park Memorial Institute 1640 (RPMI-1640) and Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution. To model psoriasis, cells were stimulated with recombinant human IL17 (Sino Biological, China) and/or TNF-α (Proteintech, USA) in serum-free medium for 24 h. To prepare HaCaT culture supernatant for culturing HUVECs, half of the medium was replaced with fresh serum-free medium after 16 h of cytokine treatments. The supernatant was collected 8 h later and centrifuged at 3,000 rpm for 10 min.
10
Breast Cancer Cell Line Maintenance
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Breast carcinoma cell lines (MCF7, T47D, HCC1806, HCC1937, BT549, MDA-MB-453, SUM149, MDA-MB-468, SKBR3, BT20, and BT549) were kindly provided by Dr. M. Götte (Department of Gynecology and Obstetrics, Münster, Germany) and Dr. B. Greve (Department of Radiation Oncology, Münster, Germany). Cells were maintained in DMEM/high glucose medium (Lonza, Basel, Switzerland) (SKBr3, MDA-MB-453, and MDA-MB-468) or in RPMI medium (MCF7, T47D, HCC1806, HCC1937, BT20, and BT549). Both media were supplemented with 10% fetal calf serum (FCS; Gibco, Waltham, MA, USA) and 100 U/mL penicillin–streptomycin (PS; Gibco). SUM149 cells were cultured in Dulbecco’s modified Eagle’s medium-F12 (1:1) with 5% FCS, insulin (5 μg/mL) (Merk, Darmstadt, Germany), and hydrocortisone (1 μg/mL; Qiagen, Hilden, Germany). MDA-MB-231 cell line was obtained from DSMZ (Leibniz Institute DSMZ–German Collection of Microorganisms and Cell Cultures) and was grown in DMEM/high glucose medium containing 10% FCS and 100 U/mL PS. HUVECs were kindly provided by Dr. D. Vestweber (Max Planck Institute of Molecular Biomedicine, Münster, Germany) or purchased from Promocell (Heidelberg, Germany) and cultured up to passage five in ECGM-2 medium supplemented with SupplementPack (PromoCell).
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