Atcc pcs 100 012

Human aortic smooth muscle cells (HSMC, ATCC-PCS-100-012) were purchased from ATCC and cultured on 0.04% gelatin-coated flasks in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin in humidified incubator supplemented with 5% CO₂. The cells were split every three days at a ratio of 1:3. Human umbilical vein ECs (HUVECs, ATCC-PCS-100-010) were cultured on 0.04% gelatin-coated flasks in M199 medium supplemented with 1 ng/ml β-EC growth factor, 3 μg/ml EC Growth Supplement from bovine neural tissue, 10 μ/ml heparin, 1.25 μg/ml thymidine, 10% FBS, 100 μ/ml penicillin and streptomycin in humidified incubator supplemented with 5% CO₂. The cells were split every three days at a ratio of 1:5. Both HSMCs and HUVECs up to passage 10 were used in this study. HEK293 and HEK293T cells were maintained in DMEM supplemented with 10% FBS and penicillin/streptomycin and were split every three days at a ratio of 1:4.

Peripheral blood mononuclear cells (PBMCs) were prepared on a Ficoll‐Hypaque density gradient centrifugation. CD14⁺ cells were obtained through positive selection by CD14⁺ micromagnetic beads according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Then, monocytes were differentiated in complete RPMI‐1640 medium supplemented with recombinant human macrophage colony‐stimulating factor (50 ng/mL; R&D Systems, Minneapolis, MN) for an additional 6 days before following study. Human primary aortic smooth muscle cells (HASMCs; ATCC PCS‐100‐012; American Type Culture Collection [ATCC], Manassas, VA) and human primary aortic endothelial cells (HAECs; ATCC PCS‐100‐011; ATCC) were cultured according to the manufacturer's instructions. Cells were used for experiments at passages 3 to 8.

Primary human aortic smooth muscle cells (hASMCs) (ATCC PCS-100-012, American Type Culture Collection, ATCC, Manassas, VA, USA) were maintained in a humidified atmosphere of 37 °C, with 5% CO₂ in VSMC basal medium (without glucose and phenol red) (ATCC^® PCS-100-030™) supplemented with recombinant human basic fibroblast growth factor (5 ng/mL), rhInsulin (5 µg/mL), recombinant human epidermal growth factor (5 ng/mL), L-glutamine (10 mM), ascorbic acid (50 µg/mL), fetal bovine serum (5%), gentamicin (10 µg/mL), penicillin (10 Units/mL), streptomycin (10 µg/mL), amphotericin B (0.28 µg/mL), and phenol red (33 µM) (ATCC). To induce clinically hyperglycemic condition, we stimulated the cells with 25 mM (450 mg/dL) of glucose. Based on our previous study for cell viability [27 (link)], we used 1 and 10 μg/mL concentration of C. turczaninowii extract in this study.

Human vascular smooth muscle cells (hVSMC; primary aortic smooth muscle cells, ATCC-PCS-100-012) and HEK 293 T cells (ATCC-CRL-11268) were purchased from American Type Culture Collection. hVSMC were cultured in vascular basal media (ATCC PCS-100-030) supplemented with the vascular smooth muscle growth kit (ATCC PCS-100-042), 100 U/mL penicillin, 100 μg/mL streptomycin. hVSMCs were used between passages 2–5. HEK 293 T cells were cultured in high-glucose Dulbecco’s Modified’s Eagle Medium containing 10 mg/mL sodium pyruvate, 2 mM L-glutamine, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The HTLA cell line, a HEK293 cell line stably expressing a tTA-dependent luciferase reporter and a β-arrestin2-TEV fusion gene^{18 (link)}, was generously provided by the laboratory of Dr. Bryan Roth and maintained in high glucose Dulbecco’s Modified’s Eagle Medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 µg/mL hygromycin B, and 2 µg/mL puromycin. All cells were cultured in a humidified environment at 37 °C, 5% CO₂.