Shields and sang m3 medium

Manufactured by Merck Group

The Shields and Sang M3 medium is a laboratory culture medium designed for the growth and maintenance of cell lines. It provides a balanced combination of nutrients, salts, and other components required for the optimal proliferation of cells in vitro.

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Lab products found in correlation

Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Hiseq 2000, by Illumina (1 mentions) Rneasy micro kit, by Qiagen (1 mentions) Colchicine, by Merck Group (1 mentions) Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions) Bacto peptone, by Merck Group (1 mentions) Shields and sang m3 insect medium, by Merck Group (1 mentions) Schneider s drosophila medium, by Thermo Fisher Scientific (1 mentions) Universal mycoplasma detection kit, by American Type Culture Collection (1 mentions) Insulin, by Merck Group (1 mentions) Centro lb 960 microplate luminometer, by Berthold Technologies (1 mentions) Dual luciferase reporter system, by Promega (1 mentions) X tremegene hp transfection reagent, by Roche (1 mentions) Insect mediasupplement, by Merck Group (1 mentions) L glutamate, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Glutamax, by Thermo Fisher Scientific (1 mentions)

7 protocols using shields and sang m3 medium

RNAi treatment of Drosophila S2 cells

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S2 cells derived from a primary culture of 20–24-h-old D. melanogaster embryos (Thermo Fisher), were cultured at 25 °C in Shields and Sang M3 medium (Sigma) supplemented with 10% heat-inactivated foetal bovine serum (Gibco). RNAi treatment was carried out according to the study by Somma et al.29 (link). Cells were suspended in serum-free Shields and Sang medium at a concentration of 1 × 10⁶ cells per ml, and plated, 0.6 ml per well, in a two-well slide culture chamber (Lab-Tek). To perform RNAi, each culture was inoculated with 5 μg ml⁻¹ of dsRNA. After 1-h incubation at 25 °C, 0.6 ml of medium supplemented with 15% foetal bovine serum was added to each well. Control cultures were prepared in the same way but without addition of dsRNA. Both RNA-treated and control cells were grown for 72 h at 25 °C and then processed for cytological analysis.

Tao L., Fasulo B., Warecki B, & Sullivan W. (2016). Tum/RacGAP functions as a switch activating the Pav/kinesin-6 motor. Nature Communications, 7, 11182.

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Transcriptome Analysis of Drosophila Hybrids

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We mated D. sechellia males to D. simulans females and dissected both oenocytes and fat bodies from the progeny, pooling material from 20 individuals from each sex. Oenocyte and fat body dissections were performed as described in [47 ]: briefly, flies were pinned to a Sylgard dissection plate (Dow-Corning) and covered with chilled Shields and Sang M3 medium (Sigma). The oenocytes and fat body of 10-day-old D. simulans/D. sechellia hybrid flies were isolated separately from the dorsal abdominal segments of both adult male and female abdomens using a fine tungsten dissecting needle. Each tissue sample represented the pooled material collected from 20 flies. Hybrid flies were reared in a 12hr light: 12 hr dark cycle and tissues dissected at equal time intervals across a 24hr period.
Immediately following dissection tissues were placed into cell lysis buffer to aid in preserving the integrity of the RNA. Total RNA was isolated using the RNeasy Micro kit (Qiagen). We collected two independent samples of each tissue from each sex, which in our experience is adequate for identifying strong patterns of allele specific expression.
We prepared libraries from the RNA using the NextFLEX RNA-seq library preparation kit (BioO Scientific, Austin, TX), and sequenced the libraries using 101bp paired end reads on an Illumina HiSeq 2000.

Combs P.A., Krupp J.J., Khosla N.M., Bua D., Petrov D.A., Levine J.D, & Fraser H.B. (2018). Tissue-specific cis-regulatory divergence implicates eloF in inhibiting interspecies mating in Drosophila. Current biology : CB, 28(24), 3969-3975.e3.

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Mitotic Arrest and Immunoblotting of Drosophila S2 Cells

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The Drosophila S2 cells were cultured at 27° in Shields and Sang M3 medium (Sigma) supplemented with 5% fetal bovine serum. To arrest cells in mitosis, S2 cells were incubated with 30 μM colchicine (Sigma) for 14–24 hr. To estimate the enrichment of metaphase stage, cells were stained with DAPI as described above. For immunoblot analysis with antibody against Pnut (Huijbregts et al. 2009 (link)), S2 cells were collected, centrifuged, and resuspended in a lysis buffer [50 mM HEPES, pH 7.6, 2.5 mM MgCl₂, 150 mM NaCl, 1 mM EDTA, 0.02% Triton X-100, 1 × Halt protease and phosphatase inhibitor cocktail (Thermo Scientific)].

Akhmetova K., Balasov M., Svitin A., Chesnokova E., Renfrow M, & Chesnokov I. (2017). Phosphorylation of Pnut in the Early Stages of Drosophila Embryo Development Affects Association of the Septin Complex with the Membrane and Is Important for Viability. G3: Genes|Genomes|Genetics, 8(1), 27-38.

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Drosophila S2 Cell Culture Protocol

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The origin of the Drosophila S2 cell line used here has been described previously [87 ]. S2 cells and S2 cells expressing tubulin-GFP were maintained at 25 °C in Shields and Sang M3 medium (Sigma) supplemented with 20% heat-inactivated fetal bovine serum (Invitrogen); all cells were free from mycoplasma contamination. The cell density was kept below 6–8 × 10⁶ cells/ml to avoid the formation of cell aggregates. cnn dsRNA production and RNAi treatments were carried out according to [4 (link)]. dsRNA-treated S2 cells were grown for 5 days at 25 °C, and then processed for TEM analysis.

Strunov A., Boldyreva L.V., Andreyeva E.N., Pavlova G.A., Popova J.V., Razuvaeva A.V., Anders A.F., Renda F., Pindyurin A.V., Gatti M, & Kiseleva E. (2018). Ultrastructural analysis of mitotic Drosophila S2 cells identifies distinctive microtubule and intracellular membrane behaviors. BMC Biology, 16, 68.

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Drosophila Cell Culture Protocols

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Cell culture was done according to the protocols established by the DGRC (Drosophila Genome Resource Center). S2-R+ cells were grown in Schneider’s Drosophila medium (Invitrogen) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin. Kc167 cells were grown in Shields and Sang M3 insect medium (Sigma) supplemented with 0.25% Bacto Peptone, 0.1% Yeast Extract, and 10% FBS. BG3-c2 cells were grown in Shields and Sang M3 medium (Sigma) supplemented with 10% FBS and 20 µg/ml insulin (Sigma). 20-hydroxyecdysone (20-HE) stimulation was done by adding 20-HE at the final concentration of 5 µM and incubating cells for the indicated times. The presence of mycoplasma in the cell lines used in this study (S2-R+, Kc167 and BG3-c2) was tested by Universal Mycoplasma Detection Kit (ATCC, #30-1012K) and no mycoplasma contamination was detected.

Zhou L., Lim M.Y., Kaur P., Saj A., Bortolamiol-Becet D., Gopal V., Tolwinski N., Tucker-Kellogg G, & Okamura K. (2018). Importance of miRNA stability and alternative primary miRNA isoforms in gene regulation during Drosophila development. eLife, 7, e38389.

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Enhancer-Blocking Assay in S2 Cells

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S2 cells were maintained in Shields and Sang M3 medium (Sigma) with 10% fetal bovine serum, BPYE and 1% Penicillin/Streptomycin. For the enhancer-blocking assay, S2 cells were transfected with 100 ng of the enhancer-blocking reporter and 250 ng of the Renilla control plasmid, using the X-tremeGENE HP transfection reagent (Roche). Cells were harvested 48 h after transfection and Luciferase activity measured using the Dual-Luciferase reporter system (Promega) with a Centro LB 960 Microplate Luminometer (Berthold).

Korenjak M., Kwon E., Morris R.T., Anderssen E., Amzallag A., Ramaswamy S, & Dyson N.J. (2014). dREAM co-operates with insulator-binding proteins and regulates expression at divergently paired genes. Nucleic Acids Research, 42(14), 8939-8953.

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Culturing Drosophila S2 and Human Cell Lines

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Cell Lines Drosophila S2 cells were cultured under 25 C in Shields and Sang M3 medium (Sigma) supplemented with 2% of fetal bovine serum (FBS, LifeTechnologies) and insect media supplement (Sigma) (Havula et al., 2013) . UT-SCC-2 cells originated from a persistent T4N1M0 Gr 2 cancer on the base of the tongue (Gre ´nman et al., 1991) . HeLa, Flp-InÔ T-RExÔ 293 cells, U2OS cells and UT-SCC-2 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma D-7777) supplemented with 10% foetal bovine serum (FBS, LifeTechnologies), L-glutamate (LifeTechnologies)/GlutaMax (LifeTechnologies), and penicillin/streptomycin (LifeTechnologies).

Liu Y., Mattila J., Ventelä S., Yadav L., Zhang W., Lamichane N., Sundström J., Kauko O., Grénman R., Varjosalo M., Westermarck J, & Hietakangas V. (2017). PWP1 Mediates Nutrient-Dependent Growth Control through Nucleolar Regulation of Ribosomal Gene Expression. Developmental cell, 43(2).

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