The mouse endothelial (SVEC4-10) cell line was obtained from American Type Culture Collection (LGC Standards GmbH, Germany). Cells were grown in Dulbecco´s modified eagle´s medium (DMEM with Glutamax; Invitrogen, Germany) supplemented with 10% heat-inactivated fetal calf serum (FCS, Invitrogen, Germany). The immortalized human micro vascular endothelial cells (HMEC-1; Centers for Disease Control and Prevention, USA) were cultured in Gibco® MCDB 131 medium supplemented with 10% (v/v) FCS, 1% (v/v) GlutaMAXTM I (100×; Life Technologies GmbH, Germany), 1 µg/mL hydrocholesterol (Sigma-Aldrich Chemie GmbH, Germany), and 10 ng/mL epidermal growth factor (Life Technologies GmbH, Germany). Mouse macrophages (J774A.1) used as control cell line were obtained from American Type Culture Collection (LGC Standards GmbH, Germany) and cultured under the same conditions as described above for the SVEC4-10 cells. All used cell lines were cultured at 37 °C in a 5% CO2 humidified environment. For experimentation, all cells were plated onto a plastic matrix at a density of 1.2 × 104 cells/cm2 (endothelial cells HMEC-1), 4.4 × 103 cells/cm2 (endothelial cells SVEC4-10) or 8.8 × 103 cells/cm2 (macrophages J774A.1). They were allowed to grow for 24 h before nanoparticle exposure. The cells were negative for mycoplasma as routinely determined via PCR.
Svec4 10
Manufactured by American Type Culture Collection
Sourced in United States, Germany
The SVEC4-10 is an endothelial cell line derived from mouse thoracic lymph node. It is a commonly used in vitro model for the study of endothelial cell biology.
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Lab products found in correlation
Hepg2, by American Type Culture Collection (6 mentions)
Renca, by American Type Culture Collection (5 mentions)
Live dead assay kit, by Thermo Fisher Scientific (4 mentions)
Gold 3 chloride trihydrate, by Merck Group (4 mentions)
L glutathione reduced, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Glutamax 1, by Thermo Fisher Scientific (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (3 mentions)
Movas, by American Type Culture Collection (3 mentions)
Fetal bovine serum (fbs), by Merck Group (3 mentions)
Sodium borohydride, by Merck Group (2 mentions)
Selenocystamine dihydrochloride, by Merck Group (2 mentions)
Hexamethylenediamine, by Merck Group (2 mentions)
Dubeccos modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Nih3t3, by American Type Culture Collection (2 mentions)
Hek293a cells, by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (2 mentions)
C57bl 6 mice, by Jackson ImmunoResearch (2 mentions)
Pea 10, by American Type Culture Collection (2 mentions)
41 protocols using svec4 10
1
Cultivation of Endothelial and Macrophage Cell Lines
2
Synthesis and Characterization of HNP-1 and Mutants
3
Cell Culture Protocols for Diverse Cell Types
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SVEC4-10 (CRL-2181; ATCC), M2 10B4 (CRL1972; ATCC), J774A.1 (TIB67; ATCC), B12 (immortalized BALB/c fibroblasts), and MEF (mouse embryonic fibroblasts from BALB/c mice) were cultivated in Dulbecco’s modified Eagle’s medium supplemented with 10 or 3% fetal calf serum. DC2.4 cells (immortalized DCs) were cultivated in RPMI medium w/o mercaptoethanol. SP2/O (CRL 1581; ATCC) cells were cultured in supplemented or plain RPMI medium. To obtain BMDCs, BMDCs were cultured for 7 d in 10% RPMI complemented with GM-CSF.
4
Murine Endothelial and Hepatoma Cell Culture
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Eight-week old male C3H mice were supplied by Charles River (Sulzfeld, Germany) and kept in the local central animal facility of the University Hospital Bonn. The mice were housed under standard conditions and had free access to water and food. Animal procedures were performed in accordance with approved protocols and followed recommendations for proper care and use of laboratory animals.
The murine endothelial cell line SVEC4-10 (ATCC CRL-2181) was obtained from LGC Promochem (Wesel, Germany) and cultured in DMEM supplemented with 10% FBS, 200 mM glutamine. HUVE-cells (HUVEC, pooled) were obtained from PromoCell (Heidelberg, Germany) and cultured in EGM with supplements according to the manufacturer's instructions.
Hepa129 cells (Hepatoma 129, obtained from NCI-Frederick Cancer Research and Development Centre (DCT Tumour Repository)) were maintained in RPMI1640 supplemented with 10% FBS, 200 mM glutamine.
The murine endothelial cell line SVEC4-10 (ATCC CRL-2181) was obtained from LGC Promochem (Wesel, Germany) and cultured in DMEM supplemented with 10% FBS, 200 mM glutamine. HUVE-cells (HUVEC, pooled) were obtained from PromoCell (Heidelberg, Germany) and cultured in EGM with supplements according to the manufacturer's instructions.
Hepa129 cells (Hepatoma 129, obtained from NCI-Frederick Cancer Research and Development Centre (DCT Tumour Repository)) were maintained in RPMI1640 supplemented with 10% FBS, 200 mM glutamine.
5
Nanomaterial Synthesis and Characterization
6
Cell Culture Protocols for Fibroblasts, Macrophages, and Endothelial Cells
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NIH3T3 murine fibroblasts (ATCC CRL-1658) were propagated in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) containing 10% heat-inactivated bovine calf serum (BCS; Life Technologies, Inc.) and 1% penicillin-streptomycin-glutamine (Life Technologies, Inc.). RAW264.7 murine macrophages (ATCC TIB-71) and SVEC4-10 endothelial cells (ATCC CRL-2181) were propagated in DMEM (Sigma) containing 10% heat-inactivated fetal calf serum (Life Technologies, Inc.) and 1% penicillin-streptomycin-glutamine (Life Technologies, Inc.).
7
Culturing Hypothalamic GnRH Neurons
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COS-7 and SVEC4-10 cells (ATCC) were grown in a monolayer at 37°C in a 5% CO2 atmosphere, in DMEM (Life Technologies, Inc.) containing 1 mM sodium pyruvate, 2 mM glutamine (Life Technologies, Inc.), 100 µg/ml streptomycin, 100 U/ml penicillin, and 4,500 mg glucose (ICN Biomedicals, Inc.), supplemented with 10% FBS (Invitrogen). The medium was replaced at 2-d intervals. Subconfluent cells were routinely harvested by trypsinization and seeded onto 58 cm2 dishes (100,000 cells). For all experiments, only cells within six passages were used.
GnV-3 cells are one of 11 clones of GnRH-expressing cells obtained by the conditional immortalization of cultured adult rat hypothalamic cells [58] (link). GnV-3 cells express markers of well-differentiated neurons and do not express markers of glial cells [59] (link). Cells were grown in Proliferation medium consisting of Neurobasal A medium with B27 supplement (20 µl/ml, Invitrogen-Gibco), PSN (1×, Invitrogen), Glutamax I (Invitrogen), doxycycline hydrochloride (0.5 µg/ml, Sigma), FBS (10 µl/ml, Biological Industries), and βFGF (5 ng/ml, Invitrogen). Doxycycline promotes the proliferation of these conditionally immortalized cells. To induce the differentiation of GnV-3 cells, the culture medium was replaced by differentiation medium (containing Neurobasal A, B27 supplement, PSN, Glutamax I, and βFGF).
GnV-3 cells are one of 11 clones of GnRH-expressing cells obtained by the conditional immortalization of cultured adult rat hypothalamic cells [58] (link). GnV-3 cells express markers of well-differentiated neurons and do not express markers of glial cells [59] (link). Cells were grown in Proliferation medium consisting of Neurobasal A medium with B27 supplement (20 µl/ml, Invitrogen-Gibco), PSN (1×, Invitrogen), Glutamax I (Invitrogen), doxycycline hydrochloride (0.5 µg/ml, Sigma), FBS (10 µl/ml, Biological Industries), and βFGF (5 ng/ml, Invitrogen). Doxycycline promotes the proliferation of these conditionally immortalized cells. To induce the differentiation of GnV-3 cells, the culture medium was replaced by differentiation medium (containing Neurobasal A, B27 supplement, PSN, Glutamax I, and βFGF).
8
Nanoparticle-based Doxorubicin Delivery
9
Murine Cytomegalovirus Mutant Generation
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NIH-3T3 (CRL-1658), SVEC4-10 (CRL-2181), HT-29 (HTB-38), were obtained from ATCC. 10.1 cells are immortalized mouse embryonic fibroblasts [41 (link)]. HEK-293A cells were purchased from Invitrogen (Karlsruhe, Germany). Cells were cultured at 37 °C and 5% CO2 in Dulbecco’s modified eagle medium (DMEM) medium supplemented with 5% or 10% fetal calf serum.
The repaired MCMV Smith strain BAC pSM3fr-MCK-2fl was kindly provided by Barbara Adler (University of Munich, Germany) [42 (link)]. MCMV mutants MCMVΔM45, MCMV-M45HA, and MCMV-M45mutRHIM have been described [43 (link)]. The MCMVΔM36 and MCMV-UL36 mutants were kindly provided by Luka Cicin-Sain (Helmholtz Centre for Infection Research, Braunschweig, Germany) [40 (link)]. MCMV-UL36 M45mutRHIM and MCMVΔM36 M45mutRHIM were generated by introducing point mutations within the M45 RHIM domain, amino acid sequence 61–64 (IQIG to AAAA), as previously described [26 (link)], into the MCMV-UL36 and MCMVΔM36 BACs by en passant mutagenesis [44 (link)]. WT and mutant MCMVs were grown and titrated on NIH-3T3 cells according to standard procedures [45 (link)]. Viral titers were determined using the median tissue culture infective dose (TCID50) method and infections were carried out with centrifugal enhancement (800× g, 30 min) when required [46 (link)].
The repaired MCMV Smith strain BAC pSM3fr-MCK-2fl was kindly provided by Barbara Adler (University of Munich, Germany) [42 (link)]. MCMV mutants MCMVΔM45, MCMV-M45HA, and MCMV-M45mutRHIM have been described [43 (link)]. The MCMVΔM36 and MCMV-UL36 mutants were kindly provided by Luka Cicin-Sain (Helmholtz Centre for Infection Research, Braunschweig, Germany) [40 (link)]. MCMV-UL36 M45mutRHIM and MCMVΔM36 M45mutRHIM were generated by introducing point mutations within the M45 RHIM domain, amino acid sequence 61–64 (IQIG to AAAA), as previously described [26 (link)], into the MCMV-UL36 and MCMVΔM36 BACs by en passant mutagenesis [44 (link)]. WT and mutant MCMVs were grown and titrated on NIH-3T3 cells according to standard procedures [45 (link)]. Viral titers were determined using the median tissue culture infective dose (TCID50) method and infections were carried out with centrifugal enhancement (800× g, 30 min) when required [46 (link)].
10
Ultrasound-Mediated Endothelial Nitric Oxide Imaging
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