Smooth muscle differentiation supplement

Manufactured by Thermo Fisher Scientific

Smooth muscle differentiation supplement is a cell culture media additive formulated to support the differentiation of smooth muscle cells from progenitor cells. It provides the necessary growth factors and signaling molecules to facilitate the transition of cells towards a smooth muscle lineage.

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5 protocols using smooth muscle differentiation supplement

Culturing Human Cell Lines for Notch1 Signaling

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Human embryonic kidney
(HEK) 293T cells stably expressing full-length Notch1 (HEK293 FLN1)
were cultured under puromycin selection (1 μg/mL) in complete
medium consisting of Dulbecco’s modified Eagle medium (Gibco)
supplemented with 5 vol % heat inactivated fetal bovine serum (Invitrogen)
and 1 vol % penicillin–streptomycin solution (Invitrogen),
at 37 °C and 5% CO₂ in a humidified atmosphere. HEK293
FLN1 were seeded at a concentration of 1.0 × 10⁵ cells/cm².
Primary human coronary artery smooth muscle cells
(HCASMC, Lonza) were purchased at passage 3 and experiments were carried
out with cells at passage 5 or 6. Cells were cultured in 231 basal
medium (Gibco) supplemented with 5 vol % smooth muscle growth supplement
(SMGS, Gibco) and 1 vol % penicillin–streptomycin solution
(Invitrogen), at 37 °C and 5% CO₂ in a humidified
incubator. As a control for the expression of α-smooth muscle
actin in the differentiated state, the basal medium was supplemented
with 1 vol % smooth muscle differentiation supplement (Gibco) instead
of SMGS. HCASMC was passaged at 80% confluency and seeded at a concentration
of 5 × 10³ cells/cm².

Putti M., Stassen O.M., Schotman M.J., Sahlgren C.M, & Dankers P.Y. (2019). Influence of the Assembly State on the Functionality of a Supramolecular Jagged1-Mimicking Peptide Additive. ACS Omega, 4(5), 8178-8187.

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Culturing Contractile and Synthetic VSMCs

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Human coronary artery smooth muscle cells (Lonza) were cultured in Human Vascular Smooth Muscle Cell Basal Medium (Gibco) supplemented with 1% penicillin/streptomycin (Sigma-Aldrich) and either 5% Smooth Muscle Growth Supplement (SMGS, Gibco) to obtain synthetic VSMCs, or 1% Smooth Muscle Differentiation Supplement (SMDS, Gibco) for a minimum of 7 days to obtain contractile VSMCs. Cells were cultured in a humidified incubator at 37°C with 5% CO₂, and media was refreshed every 2–3 days. Cells were passaged when 80%–90% confluent and used in experiments at passage 6 to 8.

Karakaya C., van Turnhout M.C., Visser V.L., Ristori T., Bouten C.V., Sahlgren C.M, & Loerakker S. (2022). Notch signaling regulates strain-mediated phenotypic switching of vascular smooth muscle cells. Frontiers in Cell and Developmental Biology, 10, 910503.

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Smooth Muscle and Cardiomyocyte Differentiation

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To evaluate the smooth muscle cell (SMC) differentiation ability, hCPCs were treated with Medium 231 (Gibco) supplemented with 5% FBS and smooth muscle differentiation supplement (Gibco) for 7 days. The medium was changed every 2 days. To investigate the cardiomyocyte differentiation ability, hCPCs were treated with MEM/EBSS medium (HyClone) supplemented with 2% FBS and 10 nM dexamethasone for 28 days. The medium was changed every day.

Park J.H., Lee N.K., Lim H.J., Ji S.T., Kim Y.J., Jang W.B., Kim D.Y., Kang S., Yun J., Ha J.S., Kim H., Lee D., Baek S.H, & Kwon S.M. (2020). Pharmacological inhibition of mTOR attenuates replicative cell senescence and improves cellular function via regulating the STAT3-PIM1 axis in human cardiac progenitor cells. Experimental & Molecular Medicine, 52(4), 615-628.

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Differentiation of hCSCs into ECs, SMCs, and CMs

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To evaluate the ability of hCSCs to differentiate into ECs, hCSCs were cultured in a DMEM high glucose medium supplemented with 20% FBS, 1% P/S, and 30 ng/mL recombinant human basic fibroblast growth factor (R&D Systems) for 7 days. To differentiate hCSCs into smooth muscle cells (SMCs), hCSCs were cultured in Medium 231 supplemented with 5% FBS, 1% P/S, and 1× smooth muscle differentiation supplement (Gibco) for 7 days. The medium was changed every 2 days. To induce cellular differentiation into CM, hCSCs were cultured in a MEM/EBSS medium (HyClone) supplemented with 2% FBS, 1% P/S, and 10 nM dexamethasone (Sigma-Aldrich) for 28 days. The medium was changed every day. The ability of cells to differentiate was analyzed by qRT-PCR and immunofluorescence.

Park J.H., Kim H., Moon H.R., Park B.W., Park J.H., Sim W.S., Kim J.J., Lim H.J., Kim Y.J., Ji S.T., Jang W.B., Rethineswaran V.K., Van L.T., Giang L.T., Yun J., Ha J.S., Ban K., Chung H.Y., Baek S.H., Park H.J, & Kwon S.M. (2021). Human cardiac stem cells rejuvenated by modulating autophagy with MHY-1685 enhance the therapeutic potential for cardiac repair. Experimental & Molecular Medicine, 53(9), 1423-1436.

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Smooth Muscle Cell Differentiation

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To measure the expression level of lncRNAs and their neighboring protein-coding genes during differentiation of smooth muscle cells, we cultured HCASM cells (Gibco) according to the manufacturer’s protocol. These cells were maintained in Medium 231 (Gibco) supplemented with Smooth Muscle Growth Supplement (Gibco), and harvested for the sample of synthetic phenotype. To obtain the sample of contractile or differentiated phenotype, HCASM cells stabilized in the growth media were cultured in Medium 231 supplemented with Smooth Muscle Differentiation Supplement (Gibco) for three days, and then collected. We used TRIzol Reagent (Invitrogen) to extract total RNA, performed reverse transcription using RevertAid Reverse Transcriptase (Thermo Scientific) with Random Hexamers (Invitrogen). Quantitative real time polymerase chain reaction (PCR) was performed using SYBR Green PCR Master Mix (Applied biosystems) in Rotor-Gene Q (Qiagen). The primer sequences for PCR were included in the supplementary table. The differentiation of these cells was confirmed by measuring the level of Calponin 1 (CNN1) and Smooth muscle protein 22-alpha (SM22α). For normalization, the expression level of Glyceraldehyde 3-phosphate dehydrogenase (GADPH) was used.

Lim Y.H., Kwon D.H., Kim J., Park W.J., Kook H, & Kim Y.K. (2018). Identification of long noncoding RNAs involved in muscle differentiation. PLoS ONE, 13(3), e0193898.

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