As previously described [19 ], primary human coronary artery smooth muscle cells (HCASMC, PromoCell, Germany) were cultured in Smooth Muscle Cell Basal Medium 2 (PromoCell), supplemented with growth medium 2 supplement pack (PromoCell) and 1% penicillin (100U/ml)/streptomycin (100 μg/ml). Cells were treated with medium containing 2 μM Zn or with medium supplemented with a Zn chelator (TPEN, 1.25 μM) or ZnCl2 (10 μM) + pyrithione (Zn ionophore, 0.5 μM). HCASMC were pre-adapted for 5d in a dual Scitive O2-controlled workstation (Baker, USA) under 18 kPa O2 (hyperoxia) or 5 kPa O2 (physiological normoxia) and 5% CO2 at 37 °C. All protocols and experiments were conducted within an O2-controlled workstation and/or plate reader (CLARIOstar, BMG Labtech, Germany) [[31] (link), [32] (link), [33] (link), [34] ].
Smooth muscle cell basal medium 2
Manufactured by PromoCell
Sourced in Germany
Smooth Muscle Cell Basal Medium 2 is a cell culture medium designed for the growth and maintenance of human smooth muscle cells. It provides the necessary nutrients and growth factors to support the proliferation and survival of smooth muscle cells in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Hcasmc, by PromoCell (2 mentions)
Clariostar, by BMG Labtech (2 mentions)
Growth medium 2 supplement pack, by PromoCell (1 mentions)
Hcaec, by PromoCell (1 mentions)
Endothelial cell growth medium mv2, by PromoCell (1 mentions)
Hek293t cell, by American Type Culture Collection (1 mentions)
Human epidermal growth factor (hegf), by PromoCell (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Fetal calf serum (fcs), by PromoCell (1 mentions)
Insulin, by PromoCell (1 mentions)
Penicillin, by Lonza (1 mentions)
Streptomycin, by Lonza (1 mentions)
Dulbecco s modified eagle s medium, by Lonza (1 mentions)
3 protocols using smooth muscle cell basal medium 2
1
Zn Modulation of HCASMC Response to O2 Levels
2
Culturing Coronary Artery Cells in Controlled Oxygen
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Primary human coronary artery endothelial cells (HCAEC) and smooth muscle cells (HCASMC) were obtained from PromoCell (Germany). HCAEC were cultured in Endothelial Cell Growth medium MV2 (PromoCell), supplemented with growth medium MV2 supplement pack (PromoCell) and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml). HCASMC were cultured in Smooth Muscle Cell Basal Medium 2 (PromoCell), supplemented with growth medium 2 supplement pack and 1% penicillin (100 U/ml)/streptomycin (100 μg/ml). HCAEC and HCASMC monolayers were maintained for at least 5 days in O2-controlled Scitive Dual or InVivo500 workstations (Baker, USA), gassed to 18, 5 or 1 kPa O2 under 5% CO2 at 37 °C. All cell culture treatments and experiments were conducted with cells within the O2-controlled workstations and/or an O2-controlled plate reader (CLARIOstar, BMG Labtech, Germany) to avoid re-exposure of cells to room air and stabilization of HIF-1α [43 (link),44 ,46 (link),47 ].
3
Generation and characterization of PXDN-KO and rescue cell lines
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HEK-293-T cells (ATCC, Manassas, VA, USA, CRL-3216), PFHR-9 cells (ATCC, Manassas, VA, USA, CRL-2423) and PXDN-KO PFHR-9 cells (prepared by Cas9 CrispR method in our laboratory earlier [21 (link)]) as well as Resc-wt and Resc-mut cells were grown in Dulbecco's Modified Eagles Medium with glutamine and 4,5 g/L glucose, supplemented with 10% fetal bovine serum (Lonza Group Ltd., Basel, Switzerland), 50 U/mL penicillin and 50 μg/mL streptomycin (Lonza Group Ltd., Basel, Switzerland) in a humidified incubator with 5% CO2 at 37 °C.
PFHR-9-Resc-wt and PFHR-9-Resc-mut cells were prepared from PXDN-KO PFHR-9 cells by transfection with the sleeping beauty transposon system [22 (link)]. Briefly, N-terminally Flag tagged WT PXDN (Resc-wt), or N-terminally Flag tagged and C-terminally V5 tagged Q823W and D826E mutant PXDN (Resc-mut) were cloned into a pSB-puro vector for transfection. Transfected cells were selected for puromycin and kept under selection (1.25 μg/mL puromycin) until one to five passages prior to experiments.
HCSMC (Lonza Group Ltd., Basel, Switzerland) and HAoSMC (kind gift of András Balla) were grown in smooth muscle cell basal medium 2 (PromoCell, Germany), supplemented with 5% fetal calf serum, 5 μg/mL insulin, 2 ng/mL hbFGF, 0.5 ng/mL hEGF (PromoCell, Germany), as well as 50 U/mL penicillin and 50 μg/mL streptomycin (Lonza Group Ltd., Basel, Switzerland).
PFHR-9-Resc-wt and PFHR-9-Resc-mut cells were prepared from PXDN-KO PFHR-9 cells by transfection with the sleeping beauty transposon system [22 (link)]. Briefly, N-terminally Flag tagged WT PXDN (Resc-wt), or N-terminally Flag tagged and C-terminally V5 tagged Q823W and D826E mutant PXDN (Resc-mut) were cloned into a pSB-puro vector for transfection. Transfected cells were selected for puromycin and kept under selection (1.25 μg/mL puromycin) until one to five passages prior to experiments.
HCSMC (Lonza Group Ltd., Basel, Switzerland) and HAoSMC (kind gift of András Balla) were grown in smooth muscle cell basal medium 2 (PromoCell, Germany), supplemented with 5% fetal calf serum, 5 μg/mL insulin, 2 ng/mL hbFGF, 0.5 ng/mL hEGF (PromoCell, Germany), as well as 50 U/mL penicillin and 50 μg/mL streptomycin (Lonza Group Ltd., Basel, Switzerland).
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