A7r5 rat aortic SMCs were obtained from American Type Culture Collection (ATCC). A7r5 cells were cultured in high glucose DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (P/S). Cells were maintained in 37°C and 5% CO2 environment. Only cells from passages 3–8 were used in experiments. For LPA treatments, A7r5 cells were quiesced in low-glucose (1 g/L) and low-FBS (0.1% FBS) media for 24 hours prior to LPA treatment. The treatment was then removed, and the cells were either immediately stimulated with 30 μM LPA for 30 minutes or cultured for an additional 1, 3, 7, or 10 days in low-glucose, low-serum media prior to LPA stimulation. Primary rat aortic SMCs were collected from 4 male Wistar rats (The Jackson Laboratory), endothelium was removed by swabbing, and the remaining tunica media was dissociated (Miltenyi Biotec tissue dissociation kit) according to the manufacturer’s instructions for 40 minutes. Subsequently, cell suspensions were forced through a 70 μm strainer washed, pooled, and cultured in a T-75 flask in DMEM with 20% FBS and 1% P/S until 80%–90% confluent. Primary human coronary artery SMCs from 3 human donors were obtained from ATCC (PCS-100-021). Human coronary artery SMCs were cultured using vascular cell basal medium (ATCC, PCS-100-030) supplemented with a VSMC growth kit (ATCC, PCS-100-042) and 20% serum to induce phenotype switching.
Pcs 100 030
Manufactured by American Type Culture Collection
Sourced in United States
The PCS-100-030 is a laboratory equipment product by American Type Culture Collection. It is a cell culture analyzer that measures the concentration and viability of cells in suspension. The device uses a specialized imaging system to count and evaluate cell samples.
Automatically generated - may contain errors
Lab products found in correlation
Pcs 100 012, by American Type Culture Collection (7 mentions)
Huvecs, by American Type Culture Collection (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Pcs 100 041, by American Type Culture Collection (3 mentions)
Pcs 100 013, by American Type Culture Collection (3 mentions)
Heracell 150i, by Thermo Fisher Scientific (3 mentions)
Pcs 100 040, by American Type Culture Collection (2 mentions)
Atcc vascular smooth muscle growth kit, by Euroclone (2 mentions)
Collagenase, by Thermo Fisher Scientific (2 mentions)
Pcs 100 042, by American Type Culture Collection (2 mentions)
Penicillin and streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Tissue dissociation kit, by Miltenyi Biotec (1 mentions)
Pcs 100 021, by American Type Culture Collection (1 mentions)
Hydrocortisone hemisuccinate, by American Type Culture Collection (1 mentions)
Ascorbic acid, by American Type Culture Collection (1 mentions)
L glutamine, by American Type Culture Collection (1 mentions)
Huvecs cc 2517, by Lonza (1 mentions)
Cc 3125, by Lonza (1 mentions)
33 protocols using pcs 100 030
1
Rat and Human Vascular Smooth Muscle Cell Culture
2
Culturing Primary Human Umbilical Vein Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were cultured and grown in vascular cell basal medium (PCS‐100‐030; ATCC) supplemented with bovine brain extract (0.2%), recombinant human (rh) EGF (5 ng/ml), L‐glutamine (10 mM), heparin sulphate (0.75 IU/ml), hydrocortisone hemisuccinate (1 μg/ml), 2% FBS and ascorbic acid (50 μg/ml). All supplements were purchased from ATCC (#PCS‐100‐040). These cells were cultured at 37°C in a CO2 humidified atmosphere and detached from the growth plates using non‐enzymatic cell dissociation solution (Cellstriper; Corning Costar).
3
Endothelial Cell Culture Protocols
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HUVECs (CC-2517) were purchased from Lonza (Basel, Switzerland) and cultured in complete endothelial basal medium (EBM) containing an endothelial cell growth medium kit (CC-3125 from Lonza). Trypsin-EDTA (0.25%) and trypsin neutralizing solution were used for HUVEC subculture. HAOECs (PCS-100-011) were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in vascular cell basal medium (PCS-100-030 from ATCC) containing an endothelial cell growth kit BBE (PCS-100-040 from ATCC). Trypsin-EDTA for primary cells (PCS-999-003 from ATCC) and trypsin neutralizing solution (PCS-999-004 from ATCC) were used for HAOEC subculture. All experiments were performed only between passages 3 and 5. All the cells were cultured in a humidified atmosphere of 5% CO2 and 21% O2 for normoxia. For hypoxic treatment, the cells were incubated in an InvivO2 400 Hypoxia Workstation chamber (RUSKINN, Bridgend, UK) at 37°C in a humidified atmosphere of 5% CO2 and 1% O2. DMOG, CoCl2, NaHS, P-(4-methoxyphenyl)-P-4-morpholinyl-phosphinodithioic acid (GYY4137), AOAA, DTT, and MG132 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Biotinylated- HIF-1α 19-mer peptide (HIF-1α, 556 to 574) and FLAG-CBS-pcDNA3 (OHu26151) plasmid obtained from GenScript (Piscataway, NJ, USA). HA-PHD2-pcDNA3 was obtained from Addgene plasmid no. 18963.
4
Isolation and Culture of Murine and Human Aortic Smooth Muscle Cells
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Murine smooth muscle cells (SMCs) were isolated from the intimal–medial layer of aortae of C57BL/6 mice of both sexes (The Jackson Lab, Bar Harbor, ME, USA) as described in [11 (link)]. Subconfluent SMCs were incubated in DMEM (Euroclone, Milan, Italy) supplemented with 0.2% essential-fatty-acid-free albumin.
Human aortic SMCs (PCS-100-012, ATCC, MA, USA) were cultured in ATCC Vascular Cell Basal Medium (PCS-100-030, ATCC; 500 mL supplemented with 500 µL ascorbic acid, 500 µL rh EGF, 500 µL rh insulin and rh FGF-b, 25 mL glutamine), 5% FBS (ATCC Vascular Smooth Muscle Growth kit), and 5 mL penicillin–streptomycin 100× (Euroclone, Milan, Italy).
Human aortic SMCs (PCS-100-012, ATCC, MA, USA) were cultured in ATCC Vascular Cell Basal Medium (PCS-100-030, ATCC; 500 mL supplemented with 500 µL ascorbic acid, 500 µL rh EGF, 500 µL rh insulin and rh FGF-b, 25 mL glutamine), 5% FBS (ATCC Vascular Smooth Muscle Growth kit), and 5 mL penicillin–streptomycin 100× (Euroclone, Milan, Italy).
5
Bioengineering 3D Tooth Bud Constructs
6
Culturing Primary Human Aortic Endothelial Cells
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Primary human aorta ECs (HAEC) were purchased from Lonza at an early passage (passages <4, #CC-2535) and cultured in Vascular Cell Basal Medium (ATCC # PCS-100–030™) supplemented with the endothelial growth media supplements (EGM™-2 # CC-4176). The HAECs were tested for mycoplasma prior to expansion and cryopreservation. 15–20% fetal bovine serum was used to maintain the cell cultures.
7
Culturing Primary Human Umbilical Vein Endothelial Cells
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Primary human umbilical vein endothelial cells (HUVECs; ATCC PCS-100-010; American Type Culture Collection; Manassas, VA, USA) were grown in an incubator with vascular cell basal medium (ATCC PCS-100-030) and endothelial cell growth kit-BBE (ATCC PCS-1-040) and 100 U/ml penicillin-streptomycin (Sigma-Aldrich, USA) under humidified atmosphere of 95% air and 5% CO2 at 37°C.[8 (link)]
8
Derivation and Culture of Primary Macrophages
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Primary mouse bone marrow-derived macrophages (BMDMs) were generated from the bone marrow of wild type and the indicated mutant mice. Cells were grown for 5–6 days in IMDM (Thermo Fisher Scientific, 12440053) supplemented with 1% non-essential amino acids (Thermo Fisher Scientific, 11140-050), 10% FBS (Biowest, S1620), 30% L929 conditioned media, and 1% penicillin and streptomycin (Thermo Fisher Scientific, 15070-063). BMDMs were then seeded into antibiotic-free media at a concentration of 1 × 106 cells into 12-well plates and incubated overnight. The human monocytic cell line THP-1 (ATCC, TIB-202) was cultured in RPMI media (Corning, 10-040-CV) supplemented with 10% FBS and 1% penicillin and streptomycin. The primary umbilical vein endothelial cells from normal human (HUVEC) (ATCC, PCS-100-013) were cultured in vascular cell basal medium (ATCC, PCS-100-030) containing cell growth factors (ATCC, PCS-100-040) and 1% penicillin and streptomycin.
9
Isolation of Human Umbilical Vein Endothelial Cells
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Umbilical cord segments (10 cm) were transported to the lab within 1 hour of delivery, and kept on ice in buffer containing PBS and antibiotics until HUVEC isolation; all isolations were performed within 2h of delivery. Umbilical segments were rinsed with PBS, and the umbilical vein was cannulated using a 3-way stopcock (Poly Medicure Limited (India), MODEL #1021). The umbilical vein was rinsed twice with 50 ml PBS before infusion with 25 ml 0.1% collagenase (Gibco, Cat. No.# 17100-017) dissolved in 37°C PBS. The cord was clamped at both ends and incubated at room temperature for 30 minutes. The cord was then unclamped and the vein flushed with vascular cell complete growth media (ATCC, PCS-100-030, containing 100 mg/dl glucose), and the effluent collected. The cells were then centrifuged and resuspended in complete growth media. Vascular cell growth media contains VEGF (5 ng/mL), EGF (5 ng/mL), FGF basic (5 ng/mL), IGF-1 (15 ng/mL), L-glutamine (10 mM), heparin sulfate (0.75 Units/mL), hydrocortisone (1 µg/mL), ascorbic acid (50 µg/mL) and fetal bovine serum (2%) (ATCC Endothelial Cell Growth Kit, PCS-100-041).
10
Modeling Endothelial Cell Responses to Amino Acid Stress
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Immortalized human umbilical vein endothelial cell line (HUVEC/TERT2) was purchased from ATCC (CRL4053, Rockville, MD, USA) and cultured in vascular cell basal medium (ATCC, PCS100030, USA) which was supplemented with endothelial cell growth kit-BBE (ATCC, PCS100040, USA). Primary human umbilical vein endothelial cell (HUVEC) was purchased from Thermo Fisher Scientific (C0035C, Waltham, MA, USA) and grown in M200 medium (Thermo Fisher Scientific, M200500, Waltham, MA, USA) containing large vessel endothelial supplement (LVES) (Thermo Fisher Scientific, A1460801, Waltham, MA, USA). For transduction, Lenti-X HEK 293T cells (Clontech, 632180, CA, USA) were cultured using DMEM high glucose medium (Gibco, USA) which was supplemented with 10% FBS and 1X Antibiotic-Antimycotic. All the cells were maintained in a humidified incubation chamber with 5% CO2 at 37 °C. Cells were treated with freshly prepared and filter sterilized Hcy (Sigma-Aldrich, H4628, USA), Cys (Sigma-Aldrich, C9768, St. Louis, MO, USA), 4-PBA (Sigma-Aldrich, P21005, USA) and TUDCA (Selleck Chemicals, S3654, Houston, TX, USA) at indicated doses. For rescue experiments, 4-PBA and TUDCA pretreatments were performed for 2 h and 14 h, respectively.
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