F0503

Manufactured by Merck Group

Sourced in United States

The F0503 is a laboratory equipment product from Merck Group. It serves as a core functional unit for various laboratory applications. The product specifications and details are maintained by the Merck Group and are not available for further interpretation or extrapolation in this response.

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Lab products found in correlation

9 protocols using f0503

Isolation and Culture of Primary Astrocytes

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Primary astrocytes were prepared from the cortex of P2 CD1 mice. Cortices were dissected in ice-cold DPBS and digested in 20 U/mL papain for approximately 10 minutes at 37°C, washed in neuronal medium and strained to remove cellular debris. Cells were plated in T25 flasks in glial medium (DMEM (FisherScientific, Cat#MT10013CV), 10% fetal bovine serum (Atlanta Biologicals Cat#S10350), 25 U/mL penicillin/streptomycin (ThermoFisher 15140122)). Cells were placed in the incubator at 37°C for 2 hours. After 2 hours, cells were washed twice with 4°C MEM (Sigma-Aldrich Cat#M2279) and media was replaced with 4°C glial medium. When flasks reached confluence, they were shaken roughly to remove all remaining non-astrocytic cells. For astrocyte and hippocampal neuron co-culture, astrocytes were plated on 96-well plates that had been coated with 40 μg/mL poly-D-lysine (PDL, Sigma-Aldrich Cat#P0899) and 1.15 mg/mL laminin (EMD Millipore Cat#CC095). Astrocytes were lifted from the flask with trypsin and plated in 96-well plates for imaging (8,500 cell/well) or 6-well plates for biochemistry (500,000 cells/well). When cells reached 70% confluence 5-fluorodeoxyuridine (5-FDU) solution (6.7 mg/mL 5-FDU (Sigma-Aldrich Cat#F0503), 16.5 mg/mL uridine (Sigma-Aldrich Cat#U3003)) was added to slow cell division.

Henderson M.X., Sedor S., McGeary I., Cornblath E.J., Peng C., Riddle D.M., Li H.L., Zhang B., Brown H.J., Olufemi M.F., Bassett D.S., Trojanowski J.Q, & Lee V.M. (2019). Glucocerebrosidase activity modulates neuronal susceptibility to pathological α-synuclein insult. Neuron, 105(5), 822-836.e7.

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Organotypic Hippocampal Slice Culture

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Organotypic hippocampal slice cultures were prepared from P5 C57BL/6 wild-type mice of both sex according to.³⁷ (link) In brief, mice were decapitated and the hippocampus was extracted and sliced into coronal sections of 300 μm thickness. The slices were placed on small pieces of Polytetrafluoroethylene (PTFE) membrane, pore size 0.45 μm (Millipore, # FHLC01300) and cultured on cell culture insert of 0.4 μm pore size (Millipore, # PICM03050) placed in a 6-well plate and then incubated at 37°C with 5% CO₂ for 18 to 23 days in vitro (DIV). The inhibitor mix containing Ara-C (Sigma-Aldrich, #C6645), Uridine (Sigma-Aldrich, #U3750) and 5-Fluoro-2′-desoxyuridine (Sigma-Aldrich #F0503), was added to a final concentration of 3 μM on the third day and the medium was exchanged three times per week.

Clavet-Fournier V., Lee C., Wegner W., Brose N., Rhee J, & Willig K.I. (2023). Pre- and postsynaptic nanostructures increase in size and complexity after induction of long-term potentiation. iScience, 27(1), 108679.

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Modulation of Hippocampal Neuron Cultures

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Timed-pregnant CD1 mice were ordered from Charles River Laboratories. Primary hippocampal neuron cultures were prepared from embryonic day 16 embryos as previously described [22 ]. At 3 days in vitro (DIV), cultures were treated with 67.5 μM 5-fluoro-2’-deoxyuridine (Sigma-Aldrich #F0503)/136 μM uridine (Sigma-Aldrich #U6381) (5-FU) to inhibit glia proliferation, allowing a nearly pure neuronal culture. At 7DIV, half of the media was removed and replaced with fresh glia-conditioned neuronal growth media containing 5-FU and either ELT (Selleck Chemicals #S2229) to a final concentration of 2, 6, or 30 μM, deferoxamine (DFO, Cayman Chemicals #14595) to a final concentration of 10 μM, or 0.2% DMSO (vehicle control) (Figure 1). Cultures were assessed daily for viability and the 30 μM ELT group was terminated at 10 DIV because of cell death. The remainder were analyzed at 14 DIV during the period of peak branching and synapse formation [23 (link)]. Postnatal glia cultures for neuronal media conditioning were prepared as previously described [22 ].

Bastian T.W., Duck K.A., Michalopoulos G.C., Chen M.J., Liu Z.J., Connor J.R., Lanier L.M., Sola-Visner M.C, & Georgieff M.K. (2017). Eltrombopag, a thrombopoietin mimetic, crosses the blood-brain-barrier and impairs iron-dependent hippocampal neuron dendrite development. Journal of thrombosis and haemostasis : JTH, 15(3), 565-574.

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Purification of Rat Dorsal Root Ganglia Neurons

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DRG from embryonic day (E) 15 rat were plated as explants on PDL-coated coverslips. After adhering within 24 h in the DMEM-HG medium (11965-084, Gibco) containing 10% FBS, cultures were maintained in serum-free neurobasal medium (NB; 21103-049, Gibco), 50 ng/ml NGF (N2513, Sigma), 2% B27 supplement (A35828-01, Gibco), and 2 mM l-glutamine (J60573.14, Gibco). After 48 h of culture, the medium was changed to DRG neuron purification medium consisting of NB medium, uridine (U6381, Sigma) and 5-fluorodeoxyuridine (F0503, Sigma) to remove non-neuronal cells. After being cycled on neuron purification medium for three times, > 95% DRG neuron purity was achieved, assessed by β-III tubulin (TuJ1, 1:500, ab18207, Abcam, Cambridge, England) staining.

Xu J., Zhang B., Cai J., Peng Q., Hu J., Askar P., Shangguan J., Su W., Zhu C., Sun H., Zhou S., Chen G., Yang X, & Gu Y. (2023). The transcription factor Stat-1 is essential for Schwann cell differentiation, myelination and myelin sheath regeneration. Molecular Medicine, 29, 79.

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C. elegans Aging Cohort Preparation

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Caenorhabditis elegans strains were maintained at 15˚C following standard culture conditions (Brenner, 1974), on NGM agar plates seeded with E. coli OP50. Aging worm cohorts were prepared as follows. Young adult hermaphrodites (30 per plate) were allowed to lay eggs for 24 hr. Several days later, L4 animals were collected and transferred to NGM/OP50 plates containing 15 µM fluorodeoxyuridine (FUDR) (Sigma‐Aldrich #F0503) to block egg production, or to NGM with 15 µM FUDR and 25 µg/ml carbenicillin (Sigma‐Aldrich #C3416), and seeded with HT115 RNAi‐producing bacteria, as described (Kamath & Ahringer, 2003), and in each case maintained at 25˚C. L4 larvae were collected in this way daily for 2–3 weeks, and then, adult hermaphrodites of each age were picked into multi‐well plates and subjected to LFASS all on the same day. For more details, see Supporting Information.

Benedetto A., Bambade T., Au C., Tullet J.M., Monkhouse J., Dang H., Cetnar K., Chan B., Cabreiro F, & Gems D. (2019). New label‐free automated survival assays reveal unexpected stress resistance patterns during C. elegans aging. Aging Cell, 18(5), e12998.

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C. elegans Lifespan Imaging Protocol

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C. elegans strains were maintained on nematode growth media (NGM) plates with Escherichia coli OP50-1 bacteria at 20°C as described by Brenner (1974) (link). SK4005: zdIs5 [Pmec-4::GFP + lin-15(+)] was used and is referred here as WT control. The daf-2(e1370) mutation was crossed into SK4005. Strains used in the experiments were maintained for at least two generations at 20°C. Synchronization was done by hypochlorite solution with direct transfer of the bleached eggs to NGM plates. C. elegans were grown to L4 at 20°C and transferred to 50 μM 5-ﬂuorodeoxyuridine (FUDR; Sigma-Aldrich F0503) plate to be maintained at 25°C (Teuscher et al., 2019 (link)). At day 1 of adulthood, ∼20 animals per condition were used for imaging, the rest were maintained to day-8 adulthood at 25°C for the second imaging session. For the experiments at 15°C, all processes are the same as above except that all C. elegans were always kept at 15°C. RNAi was performed as described in Ewald et al. (2017) (link).

Hess M., Gomariz A., Goksel O, & Ewald C.Y. (2019). In-Vivo Quantitative Image Analysis of Age-Related Morphological Changes of C. elegans Neurons Reveals a Correlation between Neurite Bending and Novel Neurite Outgrowths. eNeuro, 6(4), ENEURO.0014-19.2019.

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Caenorhabditis elegans Spaceflight Experiment

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The NIS space mission was conducted in collaboration with the Japan Aerospace Exploration Agency (JAXA). Approximately 200 synchronized L1 larvae were prepared on Day −2, added to polyethylene flight culture bags with M9 buffer with cholesterol (KH₂PO₄ 3 g, Na₂HPO₄ 12H₂O 15.1 g, NaCl 5 g, 1 M MgSO₄ 1 mL and 1 mL of cholesterol (5 mg/mL in EtOH) for a total of 1 L) and stored at 12 °C until the launch. When the samples arrived at the International Space Station (ISS), they were transferred to 20 °C incubators. The astronaut responsible for handling the samples took half of each sample and fixed them with 0.7% paraformaldehyde (PFA) when the F1-generation worms reached Day 1 and Day 10 of adulthood. To prevent starvation and the mixing of generations, the astronaut added media M9 buffer with cholesterol and E. coli food with FUdR (Sigma-Aldrich F0503, St. Louis, MO, USA, 75 μM), which interferes with DNA synthesis and prevents reproduction, after extracting the portion of samples. In this paper, only Day-10 adult samples were used for analysis. Control samples were also sent to the ISS and placed in a centrifuge that simulated a 1 G gravity force.

Kim B.S., Alcantara AV J.r., Moon J.H., Higashitani A., Higashitani N., Etheridge T., Szewczyk N.J., Deane C.S., Gaffney C.J., Higashibata A., Hashizume T., Yoon K.H, & Lee J.I. (2023). Comparative Analysis of Muscle Atrophy During Spaceflight, Nutritional Deficiency and Disuse in the Nematode Caenorhabditis elegans. International Journal of Molecular Sciences, 24(16), 12640.

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Lifespan Assay for C. elegans

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Worms were synchronized as described above using an alkaline bleach treatment to isolate embryos and cultured on e-liquid NGM agar plates for 3 days. 40 young adult nematodes were moved from each treatment plate to another plate that contained the same e-liquid supplemented with 0.1 mM FUDR (5-Fluoro-2’-deoxyuridine, F0503, Sigma-Aldrich, Saint-Louis, MO, USA). FUDR prevents the development of offspring and permits the first generation nematodes to be unambiguously identified as they age. The plates were maintained at 20 °C, with worms being moved to new, identical plates at least once to prevent starvation, and survival was assessed daily under a SMZ800 stereomicroscope (Nikon, Japan). Once per day, nematodes that didn’t actively move backward after being touched were counted as dead. Nematodes that were found dead of obvious causes unrelated to age, such as vulval rupture, were censored instead of being counted as dead.

Panitz D., Swamy H, & Nehrke K. (2015). A C. elegans model of electronic cigarette use: Physiological effects of e-liquids in nematodes. BMC Pharmacology & Toxicology, 16, 32.

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Lifespan Assay for Worms with FUdR

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L4 -stage worms synchronized from eggs were placed on NGM plates containing 100 μM 5-fluoro-2′-deoxyuridine (FUdR; F0503, Sigma-Aldrich) to prevent progeny production. The number of worms that were alive or dead was monitored and scored at regular intervals of 1 or 2 days. Worms that were ruptured, burrowed, or crawled off the plates were censored but included in the life-span analysis as censored animals. The mean life span and life span assays were analyzed using online application for the survival analysis (OASIS; http://sbi.postech.ac.kr/oasis/surv/)^{69 (link)}.

Bae E.J., Kim D.K., Kim C., Mante M., Adame A., Rockenstein E., Ulusoy A., Klinkenberg M., Jeong G.R., Bae J.R., Lee C., Lee H.J., Lee B.D., Di Monte D.A., Masliah E, & Lee S.J. (2018). LRRK2 kinase regulates α-synuclein propagation via RAB35 phosphorylation. Nature Communications, 9, 3465.

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