A549 and THP-1 cells were cultured in DMEM and RPMI1640, respectively, supplemented with 10% (fetal calf serum) FCS as described [57 (link)]. Human small airway epithelial cells (HSAEpC) were from PromoCell (Heidelberg, Germany) and were cultured in MV2 medium (Promocell, Heidelberg, Germany) as described by the manufacturer. Cells were grown until confluency with a fully supplemented medium for 48 h. For inhibitor pre-incubation, cells were washed with PBS and received serum-free medium with GI254023 (10 μM) for 30 min.
Mv2 medium
Manufactured by PromoCell
Sourced in Germany
MV2 medium is a cell culture medium formulated for the maintenance and expansion of mesenchymal stem/stromal cells (MSCs) in vitro. It is a serum-free, low-protein medium that supports the proliferation and multipotent differentiation potential of MSCs.
Automatically generated - may contain errors
Lab products found in correlation
Vascular endothelial growth factor (vegf), by PromoCell (3 mentions)
Chir99021, by Selleck Chemicals (2 mentions)
Vascular endothelial growth factor (vegf), by STEMCELL (2 mentions)
Bone morphogenetic protein 4 (bmp4), by R&D Systems (2 mentions)
Immunomagnetic microbeads, by Miltenyi Biotec (2 mentions)
Hsaepc, by PromoCell (1 mentions)
Apel2, by STEMCELL (1 mentions)
Nuclepore track etch membrane, by Cytiva (1 mentions)
Vascular endothelial growth factor (vegf), by Thermo Fisher Scientific (1 mentions)
Aprotinin, by Bayer (1 mentions)
Huvecs, by American Type Culture Collection (1 mentions)
Human epidermal growth factor (hegf), by PromoCell (1 mentions)
Basic fibroblast growth factor (bfgf), by PromoCell (1 mentions)
Ascorbic acid, by PromoCell (1 mentions)
10 protocols using mv2 medium
1
Cell Culture and Inhibitor Pretreatment
2
Directed Differentiation of hESCs into Endothelial Cells
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H9-hES-GFP clones were dissociated into single cells with Accutase, then the cells were re-suspended in the mTeSR1 medium containing 10 μM Y27632 and seeded into the lipidure-coated V-bottom 96-well plate with 9000 cells per aggregate, 150 μl per well to form EBs. On day 2, the culture medium was replaced by the mesodermal induction medium (APEL2 (STEMCELL) with 6 μM CHIR99021 (Selleck)). On day 4, the mesodermal medium was replaced by endothelial induction medium (APEL2 with 50 μg/ml VEGF (STEMCELL), 25 μg/ml BMP4 (R&D, 314-BP) and 10 μg/ml bFGF). On day 7, medium was changed into MV2 medium (Promocell) with 50 μg/ml VEGF for the maturation of endothelial cells, and the medium was renewed every other day. From day 12, the EBs were embedded into Matrigel droplets and cultured with VEGF-containing (20 μg/ml) neural differentiation medium, as that used for brain organoid culture.
3
Chemotaxis Assay for Human Endothelial Cells
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Chemotaxis of human umbilical vein endothelial cells (HUVECs, American Type Tissue Collection) was assessed using a modified Boyden chamber, as described earlier [17] (link), with 8-µm micropore polycarbonate filter (Nuclepore track-etch membrane, 155846, Whatman) coated with type 1 collagen solution at 100 ng/ml (PureColTM, 5410, Biomaterials). Cells starved overnight in 1% FCS were trypsinized and resuspended at 4.0×105/ml in MV-2 medium (Promocell GmbH), 0.25% BSA, and trasylol (Aprotinin, 495184, Bayer Healthcare) at 1,000 KIE/mL. The cell suspension was added to the upper chamber and the chemoattractant, human vascular endothelial growth factor A165 (VEGF; Peprotech), was added at 10 ng/ml to the lower chamber. HRG (100 ng/ml), iodinated with non-radioactive iodide (Merck) as described above, was added to both chambers. As a control, HRG was omitted to one set of cells. Another set of cells was incubated with HRG not exposed to the labeling procedure. After 5 h at 37°C, cells that had migrated through the filter were stained with Giemsa and counted using the Image J software (http://rsbweb.nih.gov/ij/ ). All samples were tested in at least six wells for each condition.
4
Culturing HUVECs, HAECs, and U-937 cells
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Human umbilical vein endothelial cells (HUVECs) and aortic endothelial cells (HAECs) were cultured in MV2 medium (Promocell, Heidelberg, Germany) and used for experiments between passages 3 and 6 as described previously50 (link). HEK-293 cells were cultured in DMEM, supplemented with 10% (v/v) foetal calf serum, 1 mmol/l pyruvate and 2 mmol/l L-glutamine as described previously19 (link). U-937 pro-monocytic cells were grown in RPMI 1640 medium, supplemented with 10% (v/v) foetal calf serum and 2 mmol/l L-glutamine as described previously26 (link),33 (link).
5
Isolation of Pulmonary Endothelial Cells
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Endothelial cells were isolated from the lungs of 4- to 6-week-old mice; although they were younger than mice used in our other experiments, we found that juvenile lungs produced higher-quality cell preparations for functional studies. As previously described, CD146+ pulmonary endothelial cells (PECs) were isolated with immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany) and cultured in MV2 medium (Promocell), supplemented with 10% fetal calf serum, 100 units/mL penicillin, and 100 mg/mL streptomycin (9 (link)). Cells were studied at passage 2 to 3.
6
Isolation and Characterization of Pulmonary Endothelial Cells
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Pulmonary endothelial cells (PECs) were isolated by immunoselection with CD146 antibody-coated magnetic beads as previously described and resuspended and plated in MV2 medium (Promocell), supplemented with MV2 supplement, 100 units/mL penicillin, and 100 μg/mL streptomycin (5 (link), 6 (link), 30 (link), 32 (link), 33 (link)). The endothelial cell population tested positive for a range of endothelial markers including endothelial nitric oxide synthase (eNOS), Tie2, ve-cadherin, von Willebrand factor (vWF), and CD102 protein. The cells were used from fresh isolates (P0) and were not further passaged.
7
Directed Differentiation of hESCs into ECs
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H9-hES-GFP clones were dissociated into single cells with Accutase, then the cells were resuspended in the mTeSR1 medium containing 10 μM Y27632 and seeded into the lipidure-coated V-bottom 96-well plate with 9000 cells per aggregate, 150 μl per well to form EBs. On day 2, the culture medium was replaced by the mesodermal induction medium (APEL2 [STEMCELL] with 6 μM CHIR99021 [Selleck]). On day 4, the mesodermal medium was replaced by endothelial induction medium (APEL2 with 50 ng/ml VEGF [STEMCELL], 25 ng/ml BMP4 [R&D, 314BP] and 10 ng/ml bFGF). On day 7, medium was changed into MV2 medium (PromoCell) with 50 ng/ml VEGF for the maturation of ECs, and the medium was renewed every other day. From day 12, the EBs were embedded into Matrigel droplets and cultured with VEGF-containing (20 ng/ml) neural differentiation medium, as that used for BOr culture. Detailed information about recombinant proteins and chemical compounds is available in Appendix 1—key resources table.
8
Cell Culture, Calcium Depletion, and AMPK Signaling
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HAECs and HUVECs were grown in MV2 medium (Promocell, Heidelberg, Germany) and passaged when at 80% confluence. Cells were used for experiments between passages 3 and 6 as described previously [33 (link)–35 (link)]. For experiments in which extracellular Ca2+ was depleted, cells were incubated in KRH buffer [20 mmol/l HEPES–NaOH (pH 7.4), 119 mmol/l NaCl, 5 mmol/l NaHCO3, 5 mmol/l glucose, 4.8 mmol/l KCl, 1.2 mmol/l MgSO4, 1.2 mmol/l NaH2PO4, 2.5 mmol/l CaCl2] or KRH without CaCl2 supplemented with 1 mmol/l EGTA for 2 h prior to stimulation with VEGF or AICAR. HeLa cells and HEK293 cells were cultured in DMEM supplemented with 10% (v/v) foetal calf serum (FCS). HeLa cells stably expressing LKB1 and MEFs lacking AMPKα1 and AMPKα2 (AMPK KO MEFs) were cultured as described previously [32 (link),35 (link)].
9
Isolation of CD146+ Pulmonary Endothelial Cells
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CD146+ pulmonary endothelial cells were isolated with immunomagnetic microbeads (Miltenyi) and cultured in MV2 medium (Promocell) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Cells were studied at passage 1.
10
Proliferation Dynamics of Endothelial Cells
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EC proliferation in yolk sac, hindbrain, retina, and iECs was examined using Alexa Fluor 488 and 647 Click‐iT EdU Imaging Kits (Invitrogen). 5‐ethynyl‐2′‐deoxyuridine (EdU) was prepared at 10 mg/ml in 10% dimethylsulfoxide (DMSO) for pregnant females, 5 mg/ml in 10% DMSO for pups, and for iECs, 10 μM in MV2 medium (PromoCell) with supplements (5% FCS, 5 ng/ml hEGF, 0.5 ng/ml VEGF, 20 ng/ml R3 IGF, 1 μg/μl ascorbic acid, 10 ng/ml bFGF, and 0.2 μg/μl hydrocortisone; PromoCell). Pregnant females received 200 μl EdU intraperitoneally (ip) 1 h before sacrifice, while 5 μl/gram body weight was injected ip into P6 and P7 pups 4 h before sacrifice. Embryos were collected and fixed overnight in 4% paraformaldehyde (PFA). Yolk sacs were collected and fixed for 2 h in 4% PFA. Eyes were enucleated and fixed in 4% PFA for 30 min at room temperature. iECs were incubated with EdU for 5 h and then fixed in 4% PFA. Hindbrains and retinas were dissected and immunostained to detect ERG and isolectin B4 (IsoIB4). Yolk sacs and iECs were immunostained with ERG and CD31 antibodies. Thereafter, samples were incubated for EdU detection.
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