MSCs of human umbilical cord were purchased from the ATCC (PCS-500-010) and were cultured on flasks coated with 0.04% gelatin (diluted from 2% gelatin, Sigma G1393) in ATCC mesenchymal stem cell basal medium (PCS-500-030) supplemented with one kit of mesenchymal stem cell growth kit-low serum (PCS-500-040) and 100 units/ml penicillin and streptomycin in a humidified incubator supplemented with 5% CO2. Complete MSC culture medium contained 2% FBS, 5 ng/ml rh FGF basic, 5 ng/ml rh FGF acidic, 5 ng/ml rh EGF, and 2.4 mm l -alanyl–l -glutamine. Cells were passaged every other day at the ratio of 1:2. Cells below passage 15 were used for all experiments.
Pcs 500 010
Manufactured by American Type Culture Collection
Sourced in United States, Japan
The PCS-500-010 is a laboratory equipment designed for cell culture applications. It functions as a cell culture incubator, providing a controlled environment for the growth and maintenance of cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Hucmscs, by American Type Culture Collection (1 mentions)
Hyqtase, by Thermo Fisher Scientific (1 mentions)
Carbonyl cyanide 4 trifluoromethoxy phenylhydrazone fccp, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
Hek293 cell line, by American Type Culture Collection (1 mentions)
Mesenpro rs medium, by Thermo Fisher Scientific (1 mentions)
Mesenpro growth supplement, by Thermo Fisher Scientific (1 mentions)
Y 27632, by Selleck Chemicals (1 mentions)
Glutamax, by Thermo Fisher Scientific (1 mentions)
Mtesr1 medium, by STEMCELL (1 mentions)
Matrigel, by BD (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Accutase, by STEMCELL (1 mentions)
Pcs 201 012, by American Type Culture Collection (1 mentions)
Crl 2522, by American Type Culture Collection (1 mentions)
Sodium pyruvate, by Thermo Fisher Scientific (1 mentions)
Mesencult acf medium, by STEMCELL (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
7 protocols using pcs 500 010
1
Culturing Human Umbilical Cord MSCs
2
Isolation and Culture of Cord Blood Mesenchymal Stem Cells
3
Culturing hUCMSCs and Cancer Cell Lines
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Human umbilical cord mesenchymal stem cells (hUCMSCs) were purchased from ATCC (PCS-500-010, Manassas, VA, USA). Cells were cultured at 37°C, 5% CO2 in minimal essential medium alpha, with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were passaged every 2–3 days when they reached 70–80% confluence. Passages between 5 to 12 were used in this study. Jurkat and HCT116 (purchased from Institute of Biochemistry and Cell Biology, CAS, Shanghai, China) cells were cultured with RPMI-1640, 10% FBS and 1% penicillin-streptomycin. Cells were passaged when they reached 70–80% confluence.
4
Assessing FCCP-Induced Metabolic Changes in hMSCs
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Human mesenchymal stem cells (hMSCs) isolated from Wharton’s jelly of the umbilical cord were purchased from ATCC (PCS-500-010). Cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, CA, USA) supplemented with 2% fetal bovine serum (Thermo, Logan, UT, USA) under 5% CO2 at 37 °C. Upon reaching 70–80% confluence, adherent hMSCs were detached from culture flasks by adding HyQtase (Thermo-Fischer Scientific, Waltham, MA, USA) and then seeded for cell passage or further experiments. Only hMSCs passaged 6–10 were used in our experiments.
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma Aldrich, St Louis, MO) was dissolved in DMSO at a stock concentration of 10 mM and stored at −20 °C. FCCP was used at different final concentrations of 0.1, 0.3, 1, 2, 3 μM diluted by hMSC cultured medium. Cells were exposed to different concentrations of FCCP for 20 h; then O2 consumption and cellular impedance were measured.
Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Sigma Aldrich, St Louis, MO) was dissolved in DMSO at a stock concentration of 10 mM and stored at −20 °C. FCCP was used at different final concentrations of 0.1, 0.3, 1, 2, 3 μM diluted by hMSC cultured medium. Cells were exposed to different concentrations of FCCP for 20 h; then O2 consumption and cellular impedance were measured.
5
Maintenance of HEK293, hMSCs, and hiPSCs
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The HEK293 cell line (CRL-1573, ATCC) was maintained under standard conditions in DMEM (Lonza) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMAX, and 10 mg/mL penicillin-streptomycin (all from Life Technologies). Umbilical cord blood-derived hMSCs (PCS-500-010, ATCC) were cultured in MesenPRO RS medium supplemented with MesenPRO growth supplement (Life Technologies). Cells were cultured at 37°C in a humidified atmosphere with 5% CO2 and 20% O2. hiPSCs were maintained under feeder-free conditions on Matrigel (BD Biosciences) in mTeSR1 medium (STEMCELL Technologies). Cultures were dispersed to single-cell suspensions using Accutase (STEMCELL). Cell survival before nucleofection was promoted with the Rho-associated kinase (ROCK) inhibitor Y-27632 (10 μM; Selleckchem). hiPSCs were generated in P.M.'s laboratory from human CD34+ cells from peripheral blood mononuclear cells using OKSM polycistronic SeV vectors (Muñoz-López et al., 2016 ). hiPSC research was approved by the ISCIII Steering Committee on pluripotent stem cell research.
6
Ethical Human Cell Experimentation
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The study protocol approved by the Ethical Review Committee of the Dasman Diabetes Institute (protocol No: 2013-009) and conducted in accordance with the World Medical Association Declaration of Helsinki—Ethical Principles for Medical Research Involving Human Subjects. Human WJ-MSCs, NFFs, and ASFs were purchased from the ATCC (PCS-500-010, CRL-2522, and PCS-201-012, respectively).
7
Skin Fibroblast and Stem Cell Culture
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CRL-2097 human normal skin fibroblasts (HFs) were purchased from the American Type Culture Collection (ATCC: Manassas, VA, USA). Human UCB-MSCs were purchased from the Japanese Collection of Research Bioresources cell bank (JCRB1109, Osaka, Japan) and ATCC (PCS-500-010, Manassas, VA, USA). The HFs were cultured in α-MEM (GIBCO, Carlsbad, CA, USA) fibroblast medium containing 10% fetal bovine serum, GIBCO), 1% minimum essential medium non-essential amino acid (GIBCO), 1% penicillin/streptomycin (GIBCO), and 1% sodium pyruvate (GIBCO). UCB-MSCs were cultured in MesenCult™-ACF Medium (STEMCELL technologies, Cambridge, MA, USA). The hiPSCs reprogrammed from HFs by using Sendai-virus vectors encoding Oct4, Sox2, Klf4, and c-Myc [33 (link)], or episomal vectors (Episomal iPSC reprogramming vectors, Thermo Fisher Scientific, Waltham, MA, USA) [34 (link)], were cultured with mTeSR1 (STEMCELL Technologies) medium.
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