HCASMCs were obtained from Cell Applications (San Diego, CA) at passage 3. Cells were grown in smooth muscle cell media (SMCM) supplemented with 10% heat inactivated FBS, smooth muscle cell growth supplement and 1% penicillin-streptomycin-amphotericin. Confluent HCASMCs (about 75–80%) were harvested by trypsinization and were used for isolation of protein, mRNA, and genomic DNA.
HEK-293 cells stably transfected with hSERT cDNA (HEK-hSERT) [44 (link)] were obtained from Dr. Randy Blakely (Vanderbilt University), for use as positive controls. HEK-hSERT cells from passage 15–20 were used in the experiments. The cells were cultured in high glucose DMEM (4.5 g/L of dextrose; Sigma, St. Louis, MO), with dialyzed 10% FBS (Sigma), penicillin & streptomycin (Sciencell Research Laboratories), 1% glutamine (Gibco) and 250 μg/ml geneticin (Life Technologies, Grand Island, NY). Confluent cells (75 – 80%) were harvested by trypsinization and were used for experiments.
The NCI-H727 cells (human bronchial carcinoid cell line) (ATCC, Manassas, VA) were used as cellular positive controls for TPH1 experiments. The culture media for NCI-H727 cells was ATCC-formulated RPMI-1640 Medium, with 10% heat-inactivated FBS with penicillin and streptomycin (Sciencell Research Laboratories). Confluent cells (75 – 80%) were harvested by trypsinization and used for experiments.
HEK-293 cells stably transfected with hSERT cDNA (HEK-hSERT) [44 (link)] were obtained from Dr. Randy Blakely (Vanderbilt University), for use as positive controls. HEK-hSERT cells from passage 15–20 were used in the experiments. The cells were cultured in high glucose DMEM (4.5 g/L of dextrose; Sigma, St. Louis, MO), with dialyzed 10% FBS (Sigma), penicillin & streptomycin (Sciencell Research Laboratories), 1% glutamine (Gibco) and 250 μg/ml geneticin (Life Technologies, Grand Island, NY). Confluent cells (75 – 80%) were harvested by trypsinization and were used for experiments.
The NCI-H727 cells (human bronchial carcinoid cell line) (ATCC, Manassas, VA) were used as cellular positive controls for TPH1 experiments. The culture media for NCI-H727 cells was ATCC-formulated RPMI-1640 Medium, with 10% heat-inactivated FBS with penicillin and streptomycin (Sciencell Research Laboratories). Confluent cells (75 – 80%) were harvested by trypsinization and used for experiments.