Advanced mem medium

Manufactured by Thermo Fisher Scientific

Sourced in United States

Advanced MEM medium is a cell culture medium developed by Thermo Fisher Scientific. It is formulated to support the growth and maintenance of a variety of cell types in vitro. The medium provides the necessary nutrients, vitamins, and other components required for optimal cell growth and performance.

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11 protocols using advanced mem medium

Isolation and Characterization of Swine Adipose-Derived Mesenchymal Stem Cell-Derived Extracellular Vesicles

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Mesenchymal stem cells were isolated from swine subcutaneous abdominal fat tissue, which was digested in collagenase-H, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate, as previously described [10 (link)]. At passage 3, MSCs were collected and cellular phenotype confirmed by the expression of the MSCs markers CD73, CD105, CD44, and CD90, as well as by their capacity for tri-lineage differentiation, as previously shown [16 (link), 17 (link)].
Extracellular vesicles were isolated from passage 3-MSC supernatants (10 × 10⁶ cells) by ultracentrifugation, as previously described [8 (link)–10 (link), 18 (link), 19 (link)]. Samples were centrifuged (2000g for 20 min), and the supernatant collected and subsequently centrifuged (100,000g for 1 h) at 4 °C. EVs were collected, suspended in wash buffer medium 199, centrifuged once more (100,000g for 1 h), and characterized based on the expression of common EV (CD9 and CD63) and MSC surface markers, as previously shown [8 (link), 10 (link), 20 (link)].

Meng Y., Eirin A., Zhu X.Y., O’Brien D.R., Lerman A., van Wijnen A.J, & Lerman L.O. (2018). The metabolic syndrome modifies the mRNA expression profile of extracellular vesicles derived from porcine mesenchymal stem cells. Diabetology & Metabolic Syndrome, 10, 58.

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Isolation and Characterization of MSC-Derived Extracellular Vesicles

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MSCs were collected from swine subcutaneous abdominal fat tissue (5–10g), digested in collagenase-H, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate. The third passage was collected and kept in Gibco Cell Culture Freezing Medium. Cellular phenotype was examined by the expression of the MSCs markers CD73 and CD105.
EVs were isolated from MSC supernatants (10×10⁶ cells) by the ultracentrifugation method, as previously described [4 (link)]. Samples were centrifuged (2,000g for 20min), and the supernatant collected and subsequently centrifuged (100,000g for 1h) at 4°C. EVs were collected, suspended in wash buffer medium 199, and centrifuged one more time (100,00g for 1hr).
MSCs and their daughters EVs were examined on transmission electron microscopy (JEOL 1200 EXII, Mayo Clinic’s electron microscopy core). In addition, negative staining of MSC supernatant with 2% uranyl acetate was performed to examine EV morphology.
EVs were then characterized based on the expression of both EV (CD9 and CD29) and MSC (CD73 and CD105) surface markers by western blot (AbD Serotec cat#: MCA1189 and AA120-175, and Abcam cat#: ab115289 and ab135528).

Meng Y., Eirin A., Zhu X.Y., Tang H., Chanana P., Lerman A., van Wijnen A.J, & Lerman L.O. (2017). Metabolic syndrome alters the miRNA content of porcine adipose tissue-derived mesenchymal stem cells. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 93(1), 93-103.

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Autologous Porcine Mesenchymal Stem Cell Isolation and Extracellular Vesicle Characterization

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Autologous MSCs were collected from abdominal fat (5-10g) of 4 female domestic pigs. Adipose tissue was digested in collagenase-H for 45min, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate (PLTmax, Mill Creek Life Sciences, Rochester, MN) in 37 degree/5% CO2. The 3^rd passage was collected and kept in Gibco Cell Culture Freezing Medium (Life Technologies) at -80°C for in-vitro phenotype/function analysis. We avoided the use of any animal products (beyond porcine MSCs) in our cell culture procedures to approximate a clinical-grade tissue culture product. Cellular phenotype was examined in-vitro with immuno-fluorescent staining of porcine MSCs positive for CD90, CD44, and CD105, as previously described^{5 (link), 15 (link), 16 (link)} and consistent with our experience with human MSCs^{17 , 18}.
EVs were isolated from supernatants of 10×10⁶ MSCs and cultured for 48h in advanced MEM medium without supplements. After centrifugation at 2000g, cell-free supernatants were ultra-centrifuged at 100,000g for 1h at 4°C, washed in serum-free medium containing HEPES 25mM and submitted to a second ultracentrifugation. EVs were collected and characterized based on the expression of both microvesicle (ß1-integrins, CD73, and CD40) and exosome (CD9 and CD81) markers using fluorescence activated cell sorting (FACS)^{19 (link)}.

Eirin A., Riester S.M., Zhu X.Y., Tang H., Evans J.M., O'Brien D., van Wijnen A.J, & Lerman L.O. (2014). MicroRNA and mRNA Cargo of Extracellular Vesicles from Porcine Adipose Tissue-Derived Mesenchymal Stem Cells. Gene, 551(1), 55-64.

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Isolation and Characterization of Adipose-Derived MSCs

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Cells were collected from subcutaneous abdominal fat (5–10g) using collagenase-H, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate PLTmax, Mill Creek Life Sciences, Rochester, MN). MSCs were characterized by fluorescence-activated cell sorting analysis to determine cellular phenotype for the MSC markers CD90 (abcam, ab124527) and CD105 (abcam, ab53321), as previously shown [13 (link)]. In addition, we tested that our adipose MSCs were negative for the progenitor cell marker CD34 (BD Biosciences, 340441) and the common leukocyte marker CD45 (Biolegend, 304014). The third passage was collected and kept in Gibco Cell Culture Freezing Medium for subsequent studies.

Nargesi A.A., Zhu X.Y., Hickson L.J., Conley S.M., van Wijnen A.J., Lerman L.O, & Eirin A. (2019). Metabolic syndrome modulates protein import into the mitochondria of porcine mesenchymal stem cells. Stem cell reviews, 15(3), 427-438.

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Cell Line Maintenance Protocols

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Jurkat (Weiss et al., 1984 (link)), CCRF-CEM (Foley et al., 1965 (link)), MT-1(Hinuma et al., 1981 (link)), MT-2 (Miyoshi et al., 1981 (link)), (Yamamoto et al., 1982 (link)), (Koyanagi et al., 1984 (link)), MT-4 (Koyanagi et al., 1984 (link)) , (Datta et al., 2001 (link)), C8166-45 (Salahuddin et al., 1983 (link)) and BJAB (Klein et al., 1974 (link)) were maintained in Advanced RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FCS). MET-1 cells were derived from a patient with ATL and expanded in NOD/SCID mice (Phillips et al., 2000 (link)). Vero cells (African green monkey) and Vero cells expressing CD150 (Ono et al., 2001 (link)) were grown in Advanced MEM medium (Invitrogen) supplemented with 10% FCS.

Parrula C., Fernandez S.A., Landes K., Huey D., Lairmore M, & Niewiesk S. (2014). Success of measles virotherapy in ATL depends on type I interferon secretion and responsiveness. Virus research, 0, 206-213.

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Isolation and Characterization of Swine MSCs

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MSCs collected from swine subcutaneous abdominal fat tissue (5–10g) were digested in collagenase-H, filtered, and cultured for 3 weeks in advanced MEM medium (Gibco/Invitrogen) supplemented with 5% platelet lysate(Crespo-Diaz et al., 2011 (link)). Cellular phenotype was confirmed in both groups by cell-surface marker expression(Zhu et al., 2016 (link)), and the third passage preserved in Gibco Cell Culture Freezing Medium for subsequent studies.

Meng Y., Eirin A., Zhu X.Y., Tang H., Chanana P., Lerman A., van Wijnen A.J, & Lerman L.O. (2018). Obesity-induced mitochondrial dysfunction in porcine adipose tissue-derived mesenchymal stem cells. Journal of cellular physiology, 233(8), 5926-5936.

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Syndecan Isoform Expression in K562 Cells

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Full-length SDC1–4 and SDC4 deletion mutants and transfectants, established in K562 cells (ATCC CCL-243), were created as described previously [43 (link),45 (link),46 (link)]. No His-tags were applied for the SDC constructs. Stable SDC transfectants were selected by measuring SDC expression with flow cytometry using APC-labeled SDC antibodies specific for the respective SDC isoform (all RnD Systems (Minneapolis, MN, USA); SDC1: monoclonal rat IgG1 Clone #359103, cat. no. FAB2780A [116 (link),117 (link),118 (link)]; SDC2: monoclonal rat IgG2B Clone #305515, cat. no. FAB2965A [119 (link),120 (link),121 (link)]; SDC3: polyclonal goat IgG, cat. no. FAB3539A [119 (link),122 (link)]; SDC4: monoclonal rat IgG2a clone #336304, cat. no. FAB29181A [45 (link),46 (link)]). SDC4 overexpressing A549 clones, along with WT A549 cells (ATCC CCL-185), were then grown in advanced MEM medium (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% FCS (Gibco, New York, NY, USA) at 37 °C in a humified 5% CO₂ containing air environment.

Hudák A., Letoha A., Szilák L, & Letoha T. (2021). Contribution of Syndecans to the Cellular Entry of SARS-CoV-2. International Journal of Molecular Sciences, 22(10), 5336.

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Establishing Stable SDC Transfectants in K562 and SH-SY5Y Cells

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Full-length SDC1-4 transfectants, established in K562 cells (ATCC CCL-243), were created as described previously [14 (link),16 (link),19 (link),33 (link)]. Stable SDC transfectants were selected by measuring SDC expression with flow cytometry (Becton Dickinson FACScan, Franklin Lakes, NJ, USA) using APC-labeled SDC antibodies specific to the respective SDC isoform (all RnD Systems, Minneapolis, MN, USA; SDC1: monoclonal rat IgG1 Clone #359103, cat. no. FAB2780A [66 (link),67 (link),68 (link)]; SDC2: monoclonal rat IgG2B Clone #305515, cat. no. FAB2965A [69 (link),70 (link),71 (link)]; SDC3: polyclonal goat IgG, cat. no. FAB3539A [69 (link),72 (link)]; SDC4: monoclonal rat IgG2a clone #336304, cat. no. FAB29181A [16 (link),19 (link),33 (link)]). In the case of SDC3-overexpressing SH-SY5Y cells, the transfection and selection of SDC3-overexpressing clones was carried out as described previously [19 (link)]. SDC3-overexpressing SH-SY5Y clones, along with WT SH-SY5Y cells, were then grown in Advanced MEM medium (Thermo Fischer Scientific, Waltham, MA, USA) supplemented with 10% FCS (Gibco, New York, NY, USA) at 37 °C in a humified 5% CO2 air environment. The LRP1 expression of WT K562 and SH-SY5Y cells along with SDC transfectants was measured with flow cytometry using FITC-labeled LRP1 antibodies (Santa Cruz Biotechnology, Dallas, Texas, USA, cat.no. sc-19616 FITC).

Hudák A., Jósvay K., Domonkos I., Letoha A., Szilák L, & Letoha T. (2021). The Interplay of Apoes with Syndecans in Influencing Key Cellular Events of Amyloid Pathology. International Journal of Molecular Sciences, 22(13), 7070.

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Culturing Human Melanoma Cell Lines

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Human melanoma cell line SK-MEL-28 (American Type Culture Collection (ATCC), Manassas, VA, USA) with BRAF V600E mutation was cultivated in an Advanced MEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5% fetal bovine serum (FBS, Gibco), 10 mM L-glutamine (GlutaMAX, Gibco), 100 U/ml penicillin (Grünenthal, Aachen, Germany) and 50 pg/μl gentamicin (Krka, Novo mesto, Slovenia) in a 5% CO₂ humidified incubator at 37°C.
Human melanoma cell line CHL-1 (ATCC) without BRAF V600 mutations was cultured in an Advanced RPMI 1640 medium (Gibco), supplemented with 5% FBS, 10 mM L-glutamine, 100 U/ml penicillin and 50 pg/ml gentamicin in a 5% CO ₂humidified incubator at 37°C.

Dolinsek T., Prosen L., Cemazar M., Potocnik T, & Sersa G. (2016). Electrochemotherapy with bleomycin is effective in BRAF mutated melanoma cells and interacts with BRAF inhibitors. Radiology and Oncology, 50(3), 274-279.

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HepG2 Cell Culture and Infection

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HepG2 cells were grown at 37 C and 5% carbon dioxide in Advanced MEM medium (Gibco) containing 10% heat‐inactivated fetal bovine serum, 2 mM GlutaMAX (Gibco), 1% penicillin/streptomycin (Hyclone) and a variable concentration of amphotericin B (Hyclone). Cells were seeded onto glass coverslips in 24‐well plates or glass‐bottomed culture dishes pre‐treated with rat tail collagen type I (Sigma), and allowed to grow overnight before infection with sporozoites. Media was then changed 1–2 h after infection and twice daily thereafter for all experiments.

Shears M.J., MacRae J.I., Mollard V., Goodman C.D., Sturm A., Orchard L.M., Llinás M., McConville M.J., Botté C.Y, & McFadden G.I. (2016). Characterization of the Plasmodium falciparum and P. berghei glycerol 3‐phosphate acyltransferase involved in FASII fatty acid utilization in the malaria parasite apicoplast. Cellular Microbiology, 19(1), e12633.

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