Culture media

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Culture media are liquid or solid formulations that provide nutrients and growth factors necessary for the cultivation and maintenance of cells, tissues, or microorganisms in a laboratory setting. These media are used to support the growth and proliferation of a wide range of biological specimens, including bacteria, fungi, plant cells, and animal cells.

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RPMI-1640 culture media (40 citations) Total exosome isolation from cell culture media kit (6 citations) DMEM and RPMI 1640 cell culture media (4 citations) Gibco-brand cell culture media (DMEM) (3 citations) Opti-Mem culture media (3 citations) L15 phenol red free cell culture media (2 citations) TREK para-JEM culture media (2 citations) Cell culture media and antibiotics (2 citations) Cell culture media and serum (2 citations) AIM V cell culture media (1 citations)

67 protocols using culture media

Murine Preadipocyte and Human Kidney Cell Culture

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Murine 3T3-F442A preadipocytes were provided by H. Green (Harvard University) and M. Sonenberg (Sloan-Kettering, NY). The 293T human kidney cell line was provided by M. Lazar (University of Pennsylvania) and O. MacDougald (University of Michigan). Recombinant human GH (lot # AFP8990A) for cell culture studies was purchased from the National Hormone and Pituitary Program (Torrance, CA). Recombinant human GH (Lot# N70315) for animal experiments was from Genentech (San Francisco, CA), courtesy of Dr. R.K. Menon (University of Michigan). Insulin, dexamethasone, isobutylmethylxanthine, formaldehyde, Bradford reagent, sodium orthovanadate and SYBR green were from Sigma (St. Louis, MO). Culture media, L-glutamine, and antibiotic-antimycotic were purchased from Invitrogen (Grand Island, NY). Fetal bovine serum (FBS) and calf serum (CS) were from Atlanta Biologicals (Lawrenceville, GA). Bovine serum albumin (BSA, fatty acid-free, cat. # 62150101) was purchased from Proliant (Ankeny, IA). Protease inhibitors leupeptin and aprotinin were purchased from Roche (Indianapolis, IN), and phenylmethylsulfonylfluoride (PMSF) from Mallinckrodt (St. Louis, MO).

Lin G., LaPensee C.R., Qin Z.S, & Schwartz J. (2014). Reciprocal occupancy of BCL6 and STAT5 on Growth Hormone target genes: contrasting transcriptional outcomes and promoter-specific roles of p300 and HDAC3. Molecular and cellular endocrinology, 395(0), 19-31.

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Biochemical Reagents for Cell Assays

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Fetal bovine serum (FBS) and culture media were obtained from Invitrogen (Grand Island, NY, USA). Bovine serum albumin (BSA), crystal violet, glutaraldehyde, N-(1-naphthyl) ethylenediamine (NEDA), sulfanilamide, bacterial lipopolysaccharide (LPS), SB203580 and chloroquine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acrylamide and immunoblot PVDF membranes were purchased from Bio-Rad (Madrid, Spain). BCA reagent and chemiluminescent HRP substrate were purchased from Pierce (Rockford, IL, USA). Cytokines such as TNFα, IL1β and IFNγ were obtained from Prepotech (Rocky Hill, NJ, USA). Protease inhibitors cOmplete-EDTA free were obtained from Roche (Germany). Total RNA was extracted with Trizol^® reagent (Invitrogen, Madrid, Spain) and reverse transcribed using the iScript gDNA Clear cDNA Synthesis Kit from BioRad (Madrid, Spain). qPCR was performed with iTaq Universal probes Supermix from BioRad (Madrid, Spain) in a CFX Connect Real-Time System from BioRad (Madrid, Spain). Thiobarbital was obtained from Braun Medical, S.A. (Rubí, Barcelona, Spain).

Gómez-Jaramillo L., Cano-Cano F., Sánchez-Fernández E.M., Ortiz Mellet C., García-Fernández J.M., Alcalá M., Álvarez-Gallego F., Iturregui M., González-Montelongo M.D., Campos-Caro A., Arroba A.I, & Aguilar-Diosdado M. (2022). Unravelling the Inflammatory Processes in the Early Stages of Diabetic Nephropathy and the Potential Effect of (Ss)-DS-ONJ. International Journal of Molecular Sciences, 23(15), 8450.

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Molecular Biological Reagents Protocol

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Unless otherwise noted, all chemicals, steroids, and reagents were purchased from Sigma Chemical Co. Molecular biological enzymes, pCRII-TOPO Vector, culture media, Lipofectamine RNAiMAX, siRNAs, and TRIzol reagent were purchased from Invitrogen Life Technologies, Inc. Oligonucleotide primers were purchased from Eurofins MWG operon. Antibodies were purchased from Santa Cruz Biotechnology.

Mishra B., Park J.Y., Wilson K, & Jo M. (2015). X-linked lymphocyte regulated gene 5c-like (Xlr5c-like) Is a Novel Target of Progesterone Action in Granulosa Cells of Periovulatory Rat Ovaries. Molecular and cellular endocrinology, 412, 226-238.

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Monoclonal Anti-ABCB4 Antibody Protocol

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The monoclonal P3II-26 anti-ABCB4 antibody was obtained from Enzo Life Science (Villeurbanne, France). Rabbit polyclonal anti-EBP50 was from Thermo Fisher Scientific (Rockford, IL). Rabbit polyclonal anti-Flag was obtained from Abcam (Cambridge, UK) and the monoclonal anti-β actin from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Rabbit polyclonal anti-c-myc was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Alexa Fluor-labeled secondary antibodies, DRAQ5 fluorescent probe and culture media were from Invitrogen-Life Technologies (Cergy-Pontoise, France) and peroxidase-conjugated secondary antibodies were from Rockland Immunochemicals (Gilbersville, PA). The siRNA-EBP50 duplex, the control siRNA and the ECL-Plus detection kit were from GE Healthcare France (Orsay, France). The transfection reagents Turbofect and Fugene HD were purchased from Fermentas France (Villebon-sur-Yvette, France) and Promega (Charbonnières, France), respectively. All other reagents were obtained from Sigma-Aldrich.

Venot Q., Delaunay J.L., Fouassier L., Delautier D., Falguières T., Housset C., Maurice M, & Aït-Slimane T. (2016). A PDZ-Like Motif in the Biliary Transporter ABCB4 Interacts with the Scaffold Protein EBP50 and Regulates ABCB4 Cell Surface Expression. PLoS ONE, 11(1), e0146962.

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Activation of mTOR Signaling Pathway

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Culture media were purchased from Invitrogen. Activated α₂-macroglobulin (α₂M*) was prepared as described previously [2] (link). Antibodies against mTOR, p-Akt^Thr308, p-Akt^Ser473, Akt, and PDK1 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against Raptor were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-actin antibody was from Sigma (St. Louis, MO). [³³P]-γ-ATP (specific antibody 3000 Ci/mmol) was purchased from Perkin-Elmer Life Sciences (Waltham, MA). All other materials were of analytical grade and procured locally.

Misra U.K, & Pizzo S.V. (2014). Activated α2-Macroglobulin Binding to Cell Surface GRP78 Induces T-Loop Phosphorylation of Akt1 by PDK1 in Association with Raptor. PLoS ONE, 9(2), e88373.

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Neurotrophic Properties of CX3CL1

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Recombinant human CX3CL1 was from Calbiochem/Merck (Nottingham, UK); D-serine was from Ascent Scientific (Bristol, UK); rabbit anti-phospho TrkB (Tyr 515) was from Abcam (Cambridge, UK), rabbit anti-phospo CREB (Ser 133) and rabbit anti-PARP were from Cell Signaling (Danvers, Ma, USA); secondary antibodies were from DAKO (Milan, Italy); culture media were from Invitrogen Life Technologies (San Giuliano Milanese, Italy); 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), in vitro Toxicology Assay Kit Lactic Dehydrogenase based (LDH assay), catalase, D-amino acid oxidase (DAAO), poly-L-lysine, rabbit anti-actin antibody and all the other reagents were from Sigma-Aldrich (Milan, Italy).

Lauro C., Catalano M., Di Paolo E., Chece G., de Costanzo I., Trettel F, & Limatola C. (2015). Fractalkine/CX3CL1 engages different neuroprotective responses upon selective glutamate receptor overactivation. Frontiers in Cellular Neuroscience, 8, 472.

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Cell Culture Reagents for In Vitro Experiments

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Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Atlanta, GA). Culture media, trypsin-EDTA, antibiotics, and phalloidin-Alexa488 were purchased from Invitrogen (Carlsbad, CA). Insulin, dexamethasone, and indomethacin were purchased from Sigma Aldrich (St. Louis, MO). ROCK inhibitor Y27632 was purchased from Sigma Aldrich (St. Louis, MO). Rhosin inhibitor was purchased from EMD Millipore (Billerica, MA).

Thompson W.R., Yen S.S., Uzer G., Xie Z., Sen B., Styner M., Burridge K, & Rubin J. (2017). LARG GEF and ARHGAP18 Orchestrate RhoA Activity to Control Mesenchymal Stem Cell Lineage. Bone, 107, 172-180.

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Biochemical Reagents for Cell Culture

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Enhanced chemiluminescence reagents were obtained from Amersham Biosciences (Piscataway, NJ, USA); SDS-PAGE supplies from Bio-Rad (Richmond, CA, USA); ATP, common reagents, and salts from Sigma (St. Louis, MO, USA); culture media, penicillin, streptomycin, Hanks’ balanced salt solution, L-glutamine, agarose, and RT-PCR reagents from Invitrogen (Carlsbad, CA, USA); fetal bovine serum from Hyclone (Logan, UT); Pentex bovine serum albumin (BSA, fatty acid-free, fraction V) from ICN Biomedical (Aurora, OH, USA); forskolin from Calbiochem (La Jolla, CA, USA). Krebs–Ringer bicarbonate (KRB) buffer contained (in mM) 25 HEPES (pH 7.4), 115 NaCl, 24 NaHCO₃, 5 KCl, 1 MgCl₂, and 2.5 CaCl₂.

Turk J., Song H., Wohltmann M., Frankfater C., Lei X, & Ramanadham S. (2020). Metabolic Effects of Selective Deletion of Group VIA Phospholipase A2 from Macrophages or Pancreatic Islet Beta-Cells. Biomolecules, 10(10), 1455.

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Immunoassay Reagents and Protocols

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Fetal bovine serum was purchased from Atlanta Biologicals (Atlanta,
Georgia). Culture media, trypsin-EDTA reagent, antibiotics, Lipofectamine
RNAimax, and siRNA were obtained from Invitrogen (Carlsbad, CA). RNeasy mini kit
(Qiagen, Frederick, MD) and cDNA Synthesis Kit (Bio-Rad, Hercules, California)
were used for RT-PCR. Antibodies for immunoblot and immunofluorescence were
obtained from Abcam (Cambridge, MA), Cell signaling (Danvers, Massachusetts),
Invitrogen, BD (Franklin Lakes, New Jersey), Santa Cruz (Dallas Texas), or
Jackson Immunoresearch Laboratories (West Grove, Pennsylvania) as detailed
below. Alkaline phosphatase activity kit was purchased from Abcam. Antibodies
used are shown in a table in the supplementary data set (Table S1).

Sankaran J.S., Sen B., Dudakovic A., Paradise C.R., Perdue T., Xie Z., McGrath C., Styner M., Newberg J., Uzer G., van Wijnen A.J, & Rubin J. (2019). Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells. Stem cells (Dayton, Ohio), 38(1), 102-117.

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Fetal Bovine Serum Culture Reagents

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Fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Atlanta, GA). Culture media, trypsin-EDTA, antibiotics, and phalloidin-Alexa488 were from Invitrogen (Carlsbad, CA). Ascorbic acid, and β-glycerophosphate were purchased from Sigma Aldrich (St. Louis, MO).

Thompson W.R., Uzer G., Brobst K.E., Xie Z., Sen B., Yen S.S., Styner M, & Rubin J. (2015). Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model. Scientific Reports, 5, 11049.

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