Smgm 2 singlequots

Primary human coronary artery smooth muscle cells (HCASMC) were purchased from Lonza and cultured in growth media containing SmBM Basal Medium (CC-3181, Lonza) and SmGM-2 SingleQuots supplements (CC-4149) required for growth of smooth muscle cells. THP1 monocytic cell line (DSMZ, Cat.No: ACC16) was cultured in RPMI 1640 medium supplemented with 10% (v/v) FCS, 100 U/ml penicillin and 100 mg/ml streptomycin.

The isolated uterine horns were cut open longitudinally and were washed using DPBS. The endometrial layer was removed using microscissors and discarded, whereas the myometrium was minced. The tissue pieces of myometrium were dissociated in Hank’s Balanced Salt medium containing collagenase P (1 mg/mL, from Clostridium histolyticum, Roche, Indianapolis, IN, USA), 0.2 mM CaCl₂, 0.125% bovine serum albumin (BSA) for 40 min, 37 °C. After collagenase treatment, the digested tissue pieces were gently triturated to disperse single smooth muscle cells. The obtained cells were washed with DPBS (3×) and then plated on glass coverslips. The cells were cultured in Eagle’s Minimum Essential Medium (EMEM, cat. No. 30-2003, ATCC, Manassas, VA, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin for 24–48 h in the 5% CO₂ incubator at 37 °C before the experiments. The human uterine smooth muscle cells (HUSMCs) were obtained from Lonza (CLONETICS Uterine Smooth Muscle Cell Systems, CC-2562) and cultured in the Lonza-recommended medium SmBM (Lonza,catlog No: cc-3181) supplemented with SmGM-2 SingleQuots (cat. No: cc-4149) in the 5% CO₂ incubator at 37 °C.

Human pulmonary arterial smooth muscle cells (hPASMCs) were purchased from Lonza (Walkersville, MD, USA). hPASMCs were cultured in SmGM-2 medium (Lonza) supplemented with SmGM-2 SingleQuots (Lonza) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) according to the manufacturer's instruction. The cells at passages 5–8 were used for the experiments. For the normoxic group, the cells were maintained in a humidified atmosphere of 5% CO₂ and 95% air at 37°C, while the cells from the hypoxic group were subjected to 1.5% O₂ at 37°C for 48–72 h.

Human pulmonary artery endothelial cells (hPAECs) were purchased from Lonza, Inc. (Allendale, NJ, USA). hPAECs were cultured in EBM-2 medium supplemented with 5% fetal bovine serum (FBS) supplemented with EGM-2 SingleQuots (Lonza). hPAECs were characterized by immunofluorescence with antibodies specific to vWF/Factor VIII and CD31 (PECAM). Human pulmonary artery smooth muscle cells (hPASMCs) were purchased from Lonza, Inc. (Allendale, NJ, USA) and were cultured in SmBM medium supplemented with 5% FBS and SmGM-2 SingleQuots (Lonza). hPASMCs were characterized by immunofluorescence with antibodies specific to α-smooth muscle actin. Additionally, hPAECs and hPASMCs were further characterized by morphological observation throughout the serial passages and were only used between passages 2 through 7 for this study. Cells were grown in 5% CO₂ at 37 °C and were passaged at the confluence. All of the cells tested negative for mycoplasma, bacteria, yeast, fungi, HIV-1, hepatitis B, and hepatitis C before use.