Packaging cell line 293GP, TIME cells, and human AML cell line HEL-2 were purchased from ATCC. MOLM-13, NOMO-1, and OCI-AML3 were purchased from DSMZ. THP-1 cells stably transduced with GFP-luciferase were kindly provided by Dr. Terry Fry at NIH (Bethesda, MD). iHUVECs were a gift from Dr. David Sullivan at Northwestern University (Chicago, IL). iHUVEC-19 cells were generated by transducing iHUVECs with a lentivirus containing a truncated version of CD19, including the extracellular and transmembrane domain only. Transduced cells underwent flow cytometry–based sorting on a BD FACSAria II to isolate a uniform population of CD19+ iHUVECs. All cell lines were verified by short tandem repeat analysis and confirmed to be Mycoplasma negative by PCR.
Time cells
Manufactured by American Type Culture Collection
Sourced in Japan, United States
TIME cells are a human endothelial cell line derived from human microvascular endothelial cells. They exhibit characteristics of endothelial cells and can be used for in vitro studies of endothelial cell biology.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Vascular cell basal medium, by American Type Culture Collection (2 mentions)
Human anti scarf1, by R&D Systems (2 mentions)
Maf2409, by R&D Systems (2 mentions)
Microvascular endothelial cell growth kit vegf, by American Type Culture Collection (2 mentions)
Facsaria 2, by BD (1 mentions)
Molm 13, by Leibniz Institute DSMZ (1 mentions)
Oci aml3, by Leibniz Institute DSMZ (1 mentions)
Nomo 1, by Leibniz Institute DSMZ (1 mentions)
Blasticidine, by Merck Group (1 mentions)
Microvascular endothelial cell growth kit vegf, by Merck Group (1 mentions)
Hmvec, by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Hek293tn cells, by System Biosciences (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (1 mentions)
Ea hy926, by American Type Culture Collection (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
8 protocols using time cells
1
Cell Line Characterization and Transduction
2
Detailed Cell Culture Protocol
3
Immortalized Endothelial Cell Culture
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Human telomerase (hTERT)-immortalized microvascular endothelial cells isolated from human foreskin (TIME cells; ATCC) were grown in ATCC’s vascular cell basal medium supplemented with microvascular endothelial cell growth kit-VEGF and 12.5 μg/ml blasticidine (Sigma-Aldrich) for 48 h at 37°C + 5% CO2 until reaching 80–90% confluence. At confluence, cells were passaged and 5.4 × 104 cells were plated on fibronectin-coated (10 µg/ml; MilliporeSigma), base/acid cleaned, 0.17 mm (#1.5) glass-bottom dishes (14 mm glass diameter; MatTek) for 18 h prior to experiments.
4
Culturing HMVECs, TIME, and HEK293TN cells
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HMVECs (Cascade Biologics Inc.) and TIME cells (#CRL4025, ATCC) were maintained in HuMedia-EB2 (HMEB2) medium supplemented with 5% FBS, 5 ng/mL basic fibroblast growth factor, 10 μg/mL heparin, 10 ng/mL epidermal growth factor, 1 μg/mL hydrocortisone, 39.3 μg/mL (80 µM) dibutyryl cAMP, 50 µg/mL gentamicin and 50 ng/mL amphotericin B (growth medium) according to the manufacturer’s instructions (Kurabo Corp., Tokyo Japan). HEK293TN cells (System Biosciences) were maintained in DMEM (Sigma-Aldrich) supplemented with 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin.
5
Cytotoxicity Assay of TIME Cells
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Reagents were purchased from Sigma-Aldrich unless otherwise stated. Complete media consisted of RPMI-1640 (Gibco, Thermo-Fisher, Grand Island, NY) supplemented with 100 U/mL penicillin, 100 U/mL streptomycin, 2 mM Glutamax and 10% fetal bovine serum (FBS, Hyclone GE Lifescience, Pittsburgh, PA). TIME cells (Cat#: CRL-4025) were obtained from the ATCC (Manassas, VA). Culture of the cell line required Vascular Cell Basal Medium (ATCC, Cat#: PCS-100-030), supplemented with microvascular endothelial cell growth kit-VEGF (ATCC, Cat#: PCS-110-041). Mouse embryonic fibroblasts (MEFs; Cat# SCRC-1008) were obtained from the ATCC. Flow cytometry antibodies were obtained from BD Biosciences. Human anti-SCARF1 (Cat#: AF2409 and MAF2409) antibodies were purchased from R&D Systems.
6
Culturing Endothelial Cell Lines
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HUVECs and telomerase-immortalized human microvascular endothelium cell line (TIME cells) were purchased from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained in complete ECM medium (Catalog #1001, ScienCell, Carlsbad, CA, USA) supplemented with 5% FBS, the Endothelial Cell Growth Supplement, penicillin (100 U/ml), and streptomycin (100 μg/ml) as described.66
7
Cultivation and Serum Starvation of Cell Lines
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HEK-293 cells, A7r5, EA.hy926 and TIME cells (American Type Culture Collection) were cultivated in the recommend media as indicated in Supplemental Table S2 and cultivated 37 °C with 5% CO2. 24 h prior to the experiments, cells were cultivated in serum-free medium for 24 h and treated with vehicle (DMSO 0.1%), aldosterone or a cytokine mixture composed of IL-1β (10 ng/ml), IL-6 (20 ng/ml), TNFα (20 ng/ml). Single exception represents the TIMEs which made quiescent by utilization of charcoal-stripped steroid-free medium with a reduced FCS content (2%) for 24 h.
8
Culturing Diverse Cell Lines for Research
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MMNK‐1 cells (normal cholangiocytes), normal human dermal fibroblast (NHDF) cells and human cancer cell lines were prepared as previously described.14 , 15 Briefly, cells were maintained in RPMI 1640 containing 10% FBS with 100 unit/mL penicillin and 100 mg/mL streptomycin (Life Technologies, Tokyo, Japan) at 37°C with 5% CO2. NuLi‐1 cells (normal human bronchus cells), TIME cells (human microvascular endothelium cells) and human pancreatic duct epithelial cell (HPNE) cells were purchased from the American Type Culture Collection (ATCC) and cultured following the instructions. A hepatocellular carcinoma line, HAK‐1B, and a lung adenocarcinoma line, PC‐9, were kind gifts from Dr Yano (Department of Pathology, Kurume University) and Dr Kiura (Department of Respiratory Medicine, Okayama University), respectively.
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