ASC52telo, native or CRISPR/Cas9-modified, were cultured under standard conditions until reaching 90% confluence. Afterwards, the cells were washed three times with Hank’s solution (PanEco, Moscow, Russia, #P020п) and incubated for 48 h in DMEM low-glucose medium (ThermoFischer Scientific, #11885084) supplemented with 1% antibiotic–antimycotic (Gibco). Then, the obtained medium was centrifuged for 10 min at 300 g to remove cell debris. The supernatant containing EVs was decanted and concentrated to 5-fold using Amicon centricons (300 kDa, Sartorius, Göttingen, Germany, #Vs3052). The concentrated conditioned medium enriched in EV fractions was used in the cellular model of fibrosis or stored at −80 °C.
Dmem low glucose medium
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom, China
DMEM low-glucose medium is a cell culture medium formulation used for the in vitro cultivation of various cell types. The medium contains a reduced concentration of glucose compared to standard DMEM formulations. The core function of this product is to provide a balanced nutrient solution to support the growth and maintenance of cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (24 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (8 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
Glutamax, by Thermo Fisher Scientific (6 mentions)
Penicillin, by Thermo Fisher Scientific (6 mentions)
Trypsin edta, by Thermo Fisher Scientific (5 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (5 mentions)
Pen strep, by Thermo Fisher Scientific (4 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (4 mentions)
Trypsin inhibitor, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Collagenase p, by Roche (3 mentions)
L glutamine, by Thermo Fisher Scientific (3 mentions)
Non essential amino acid solution, by Merck Group (2 mentions)
Ca2 mg2 free phosphate buffered saline, by Thermo Fisher Scientific (2 mentions)
Gene pulser 2 electroporation system, by Bio-Rad (2 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions)
71 protocols using dmem low glucose medium
1
Isolation of Extracellular Vesicles from Cell Culture
2
Detailed Cell Culture Conditions for Diverse Cancer Cell Lines
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Human total RNA from different tissues used in this study was purchased to Takara Bio/Clontech (Shiga, Japan).
MCF7, T47D, BT474, MDA-MB-231, MDA-MB-453, MDA-MB-468, RCC4, 786-0, U2OS mTUNE, and human fibroblasts were cultured in DMEM low glucose medium (1g/L; Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% FBS no longer than 20 passages. HUVEC cells were purchased from Lonza (Basilea, Switzerland) and grown in EGM2 medium for maximum 7 passages. They were all mycoplasma tested every 3 months and authenticated during the course of this project. Cells were subjected to 1% or 0.1% hypoxia for the periods specified in each experiment using an InVivO2 chamber (Baker, Sandford, Maine).
U2OS mTUNE glioblastoma cell lines were kindly donated by Dr Christian Frezza. Three isogenic cell lines with different levels of heteroplasmy of mutated mtDNA were used: M7, M45 and M80 (24 (link)).
143B and 143B Rho Zero (Rho Zero) cells were a gift of Dr Karl Morten. Rho Zero cells were generated by treating 143B cells (TK1 deficient) with 10µM 2’,3’-dideoxycytidine (ddC) for 10 days. Both cell lines were grown in high glucose DMEM (Gibco, Carlsbad, California) supplemented with 10% FBS. Rho Zero cell culture media was additionally supplemented with 50µg/mL uridine (A15227.06, Alfa Aesar, Haverhill, Massachusetts).
MCF7, T47D, BT474, MDA-MB-231, MDA-MB-453, MDA-MB-468, RCC4, 786-0, U2OS mTUNE, and human fibroblasts were cultured in DMEM low glucose medium (1g/L; Thermo Fisher Scientific, Waltham, Massachusetts) supplemented with 10% FBS no longer than 20 passages. HUVEC cells were purchased from Lonza (Basilea, Switzerland) and grown in EGM2 medium for maximum 7 passages. They were all mycoplasma tested every 3 months and authenticated during the course of this project. Cells were subjected to 1% or 0.1% hypoxia for the periods specified in each experiment using an InVivO2 chamber (Baker, Sandford, Maine).
U2OS mTUNE glioblastoma cell lines were kindly donated by Dr Christian Frezza. Three isogenic cell lines with different levels of heteroplasmy of mutated mtDNA were used: M7, M45 and M80 (24 (link)).
143B and 143B Rho Zero (Rho Zero) cells were a gift of Dr Karl Morten. Rho Zero cells were generated by treating 143B cells (TK1 deficient) with 10µM 2’,3’-dideoxycytidine (ddC) for 10 days. Both cell lines were grown in high glucose DMEM (Gibco, Carlsbad, California) supplemented with 10% FBS. Rho Zero cell culture media was additionally supplemented with 50µg/mL uridine (A15227.06, Alfa Aesar, Haverhill, Massachusetts).
3
Culturing Macrophages, Monocytes, and Mesenchymal Stromal Cells
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The mouse M0 macrophage, RAW264.7 cells (Cat# TIB-74), and human pro-monocyte, U937 cells (Cat# CRL-1593.2) were purchased from the American Type Culture Collection (ATCC). The human turbinate mesenchymal stromal cell line (hMSCs), harvested by Kwon et al. [32 (link)], was also used in this study. The RAW264.7 cells were cultured in DMEM high glucose medium supplemented with 3.72 mg/ml NaHCO3, 5.685 mg/ml, HEPES, 10% heat activated fetal bovine serum (FBS), 2 mM l -glutamine, 0.25 μg/ml amphotericin B, and 100U/ml penicillin-streptomycin (P/S). The U-937 cells were cultured and maintained in RMPI medium 1640 (1X) (Thermo Fisher Scientific) supplemented with 10% FBS and 100U/ml P/S. Both the RAW264.7 and U-937 cells were passaged every 3 days. The hMSCs were cultured for 4–5 days in DMEM low glucose medium (Thermo Fisher Scientific) supplemented with 10% FBS and 100U/ml P/S. The cell culture medium was replaced every 3 days and the cells were passaged at 80% confluence. All the cells were kept in the humidified incubators at 37 °C and 5% CO2.
4
HepG2 Cytotoxicity Assay with Resazurin
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For the cytotoxicity assay, HepG2 cells were seeded in 96-well plates and grown in DMEM low glucose medium (ThermoFisher, 10567014) supplemented with 10% FBS. Test compounds were added in different concentrations, with a final concentration of 1% DMSO and cells were incubated for 20 h at 37°C with 5% CO2. Next, resazurin was added to reach a final concentration of 0.1 mM. After 3 h incubation, the fluorescence was measured on a PHERAstar microplate reader (BMG Labtech) using an excitation wavelength of 540 nm and emission wavelength of 590 nm. The average intensity of DMSO control was set as 100% alive and the percentage of intensity from each treated sample was calculated. IC50 was calculated using non-linear regression in GraphPad Prism. Experiments were conducted in biological triplicates.
5
Isolation and Culture of Human Bone Marrow-Derived Mesenchymal Stem Cells
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Bone marrow aspirates were obtained by percutaneous direct aspiration from the iliac crest of 5 healthy volunteers at University Hospital Virgen de la Arrixaca (Murcia, Spain). Bone marrow was collected with 20 U/ml sodium heparin, followed by a Ficoll density gradient-based separation by centrifugation at 540 g for 20 min. After, mononuclear cell fraction was collected, washed twice with Ca2+/Mg2+-free phosphate buffered saline (PBS) (Gibco Invitrogen) and seeded into 175-cm2 culture flasks (Nunc, Thermo Fisher Scientific) at a cell density 1.5 × 105 cells/cm2 in serum-containing media, composed of DMEM low glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Lonza), 1% GlutaMAX (Thermo Fisher Scientific), non-essential amino acid solution (Sigma-Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). After 3 days of culture at 37 °C and 7% CO2, non-attached cells were removed and fresh complete medium was added. Culture media were renewed every 2 days, and the isolated hBMSCs were passaged when cultures were 70–80% confluent. All studies were performed using hBMSCs expanded within culture passages 3–4.
6
Transfection of hBMSCs for Neural Differentiation
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The expression vectors used in the present study were H2B-eGFP, a gift from Geoff Wahl (Addgene plasmid # 11680; http://n2t.net/addgene:11680 ; RRID:Addgene_11680; Kanda et al.67 (link)). Isolated hBMSCs-derived cells were transfected using the Gene Pulser-II Electroporation System (Bio-Rad Laboratories). Electroporation was performed in a sterile cuvette with a 0.4-cm electrode gap (Bio-Rad Laboratories), using a single pulse of 270 V, 500 μF. Plasmid DNA (5 μg) was added to 1.5 × 106 viable hBMSCs-derived cells in 0.2-ml DMEM low glucose medium (Thermo Fisher Scientific) before electrical pulsing. hBM-MSCs-derived cells were transfected 1 day before neural differentiation.
7
Equine ASC Exosome Isolation
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Equine ASC were cultured in exosome-depleted medium which was obtained by ultracentrifugation of a DMEM low glucose medium (Thermo Fisher Scientific, Germany) in the presence of 5% FCS (Biosell, Germany) at 100.000 g over night at 4 °C. Near confluent cells (80–90%) were washed with 20 ml of DMEM low glucose without FCS on a shaker and were incubated in a humified incubator (37 °C in 5% CO2) in 10 ml DMEM low glucose with supplementation of 1x ITS (Insulin Transferrin-Selenium, Thermo Fisher Scientific) for 48–72 h. Supernatant was collected and centrifuged at 2.700 g for 5 min and afterwards filtered through a 0.2 μm filter (Sarstedt, Germany). Exosomes were isolated from this filtrate with three different isolation procedures.
8
Investigating UV-Induced Stress Response in MCF7 Cells
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MCF7 human breast carcinoma cells were maintained in Dulbecco's modified Eagle's Medium (Thermo Fisher Scientific, 12100-046) with 10% FBS (Thermo Fisher Scientific, 10500-064) and 1% Pen-Strep (Thermo Fisher Scientific, 15140-122). Cells were exposed to 10 J/m2 short wavelength UV (UVC) irradiation in UVC crosslinker (CL 1000, UVP). Cells were transfected with different vectors, siRNAs (siGENOME SMART pool, ELAVL1 (M-003773-04-0005), p53(M-003329-03) and Non Targeting siRNA pool (D-001206-13-05) GE-Dharmacon) and antagomiR against miR-125b (Trilink Biotechnologies, C33-B02A) using Lipofectamine 2000 (Thermo Fisher, 11668019) in DMEM low glucose medium (Thermo Fisher, SH30021.01). DNA amount for transfection was equalised by pGEMT plasmid (Promega, A3600).
9
Isolation and Expansion of hMB-MSCs
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A standard protocol for the isolation and expansion of hMB-MSCs was used as previously described [40 (link)]. Bone marrow aspirates were obtained by percutaneous direct aspiration from the iliac crest of 5 healthy volunteers at University Hospital Virgen de la Arrixaca (Murcia, Spain). Bone marrow was collected with 20 U/ml sodium heparin, followed by Ficoll density gradient-based separation by centrifugation at 540 g for 20 min. After, the mononuclear cell fraction was collected, washed twice with Ca2+/Mg2+-free phosphate buffered saline (PBS) (Gibco Invitrogen) and seeded into 175-cm2 culture flasks (Nunc, Thermo Fisher Scientific) at a cell density of 1.5 × 105 cells/cm2 in serum-containing media (designated as the basal media), composed of DMEM low glucose medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Lonza), 1% GlutaMAX (Thermo Fisher Scientific), nonessential amino acid solution (Sigma‒Aldrich) and 1% penicillin/streptomycin (Thermo Fisher Scientific). After 3 days of culture at 37 °C and 7% CO2, nonadherent cells were removed, and fresh complete medium was added. Culture media were renewed every 2 days, and the isolated hMB-MSCs were passaged when cultures were 70–80% confluent. All studies were performed using hMB-MSCs expanded within culture passages 3–4.
10
Quantifying Glucose Uptake in Adipocytes
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Differentiated WT and SSAO−/− adipocytes cultured in a 24-well plate were labeled overnight with the trace levels (0.3 μ Ci/well) of [14C] glucose (Perkin Elmer) in DMEM low-glucose medium (1 g/L, ThermoFisher) with 10% FBS (Sigma Aldrich). Lipids were extracted with chloroform/methanol using the Folch method [43 (link)], and radioactivity was counted by liquid scintillation. Cell seeded in a 24-well plate at the same time were lysed with 1x RiPA buffer for protein content analysis using BCA assay kit (Pierce). Radioactivity of cell lysates was counted in Ecoscint scintillation mixture (National Diagnostics) using a Beckman LS-6000 liquid scintillation counter. Radioactivity in each sample was normalized to protein content.
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