D1145

Manufactured by Merck Group

Sourced in United States

D1145 is a laboratory equipment product offered by Merck Group. It is designed for general laboratory use, providing a reliable and consistent performance for various experimental and analytical applications. The core function of D1145 is to facilitate standard laboratory procedures without additional interpretation or extrapolation.

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Lab products found in correlation

Trypan blue, by Merck Group (1 mentions) Mcf 7, by Merck Group (1 mentions) P4333, by Merck Group (1 mentions) F6765, by Merck Group (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Hepes, by Thermo Fisher Scientific (1 mentions) Ack lysing buffer, by Lonza (1 mentions) A5955, by Merck Group (1 mentions) P211107, by PAN Biotech (1 mentions) Transwell chamber, by BD (1 mentions) 4 kda fitc dextran, by Merck Group (1 mentions)

9 protocols using d1145

Zinc-Induced MDCK II Cell Imaging

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A detailed protocol for ZnUMBA using MDCK II cells will be published elsewhere (Higashi et al., 2022 (link)
Preprint). Briefly, the cells were seeded onto the bottom surfaces of 12-well polycarbonate Transwell filters with 0.4-µm pore size (#3401; Corning) at a density of 1.0 × 10⁵ cells/cm² and cultured until TER reached a plateau. Before imaging, both the apical and basal sides of the cells were washed twice with Z medium (30% HBSS, 65% phenol red-free DMEM [D1145; Sigma-Aldrich], and 5% FBS). The filter was placed on a glass-bottom dish containing 2 mM of ZnCl₂ in Z medium, which faced the apical side of the cells. 10 µM of FluoZin-3 and 1 µM of Ca-EDTA in Z medium were placed in the filter, which faced the basal side of the cells. The cells were imaged using a confocal microscope, as described above. At the end of the imaging, the addition of excess ZnCl₂ to the basal medium helped in the identification of the cell borders.

Higashi T., Saito A.C., Fukazawa Y., Furuse M., Higashi A.Y., Ono M, & Chiba H. (2022). EpCAM proteolysis and release of complexed claudin-7 repair and maintain the tight junction barrier. The Journal of Cell Biology, 222(1), e202204079.

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Culturing ER+ Breast Cancer Cell Lines

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MCF-7 and T-47D, two human ER (+) breast carcinoma cell lines were used for this study. Both cell lines were purchased from American Type Culture Collection (ATCC). MCF-7 cells were maintained in DMEM high glucose media with l-Glutamine (D5796, Sigma-Aldrich) supplemented with 10% heat-inactivated fetal bovine serum (FBS; F6765, Sigma-Aldrich) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich). T-47D cells were maintained in RPMI medium 1640 with l-Glutamine (11875–093, ThermoFisher Scientific) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin. RPMI and DMEM high glucose media without l-Glutamine, sodium pyruvate and phenol red (D1145, Sigma-Aldrich) supplemented with 10% FBS charcoal-stripped (F6765, Sigma-Aldrich) and 1% penicillin/streptomycin (P4333, Sigma-Aldrich) were used for the experimental conditions of MCF-7 and T-47D cells. All cells were cultured at 37 °C in a 5% CO₂ humidified environment. Cultures were periodically verified and confirmed to be free of mycoplasma. Passages were performed at 75–80% confluence using 0.5% trypsin (59418C, Sigma-Aldrich). Cell lines were used below 20 passages. Viable cells were identified using the Trypan Blue (T8154, Sigma-Aldrich) exclusion method and counted using a hemocytometer.

Reyes-Ramos A.M., Álvarez-García Y.R., Solodin N., Almodovar J., Alarid E.T., Torres-Garcia W, & Domenech M. (2021). Collagen I Fibrous Substrates Modulate the Proliferation and Secretome of Estrogen Receptor-Positive Breast Tumor Cells in a Hormone-Restricted Microenvironment. ACS biomaterials science & engineering, 7(6), 2430-2443.

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ChIP-seq sample preparation protocol for Ishikawa and JHUEM-14 cells

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Ishikawa and JHUEM-14 cells were plated on to 10-cm tissue culture dishes in phenol red-free DMEM (Sigma-Aldrich #D1145) supplemented with l-glutamine, sodium pyruvate, and 10% charcoal-dextran-stripped FBS. Three days later, media were replaced and cells incubated with fresh medium containing either 10 nM estradiol or DMSO (vehicle control) for 3 h. Cells were washed twice with PBS and fixed at room temperature in 1% formaldehyde in PBS. After 10 min, cells were placed on ice and washed twice with ice-cold PBS. The reaction was quenched with 10 mM DTT in 100 mM Tris-HCl (pH 9.4) and cells removed from the dish with a cell scraper. Cells were incubated at 30 °C for 15 min, then spun for 5 min at 2000×g. Cells were washed sequentially with ice-cold PBS, buffer I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5) and buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5) and centrifuged for 5 min at 2000×g at 4 °C. Cells were resuspended in 300–750 μl of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, with complete protease inhibitor cocktail (Sigma-Aldrich #11836145001)). Ishikawa cells were sonicated for eight cycles (10 s) and JHUEM-14 cells for 20 cycles using the highest power setting of a Branson Digital Sonifier SLPt. After chromatin shearing was confirmed by agarose gel electrophoresis, samples were centrifuged for 10 min at 4 °C.

O’Mara T.A., Glubb D.M., Amant F., Annibali D., Ashton K., Attia J., Auer P.L., Beckmann M.W., Black A., Bolla M.K., Brauch H., Brenner H., Brinton L., Buchanan D.D., Burwinkel B., Chang-Claude J., Chanock S.J., Chen C., Chen M.M., Cheng T.H., Clarke C.L., Clendenning M., Cook L.S., Couch F.J., Cox A., Crous-Bous M., Czene K., Day F., Dennis J., Depreeuw J., Doherty J.A., Dörk T., Dowdy S.C., Dürst M., Ekici A.B., Fasching P.A., Fridley B.L., Friedenreich C.M., Fritschi L., Fung J., García-Closas M., Gaudet M.M., Giles G.G., Goode E.L., Gorman M., Haiman C.A., Hall P., Hankison S.E., Healey C.S., Hein A., Hillemanns P., Hodgson S., Hoivik E.A., Holliday E.G., Hopper J.L., Hunter D.J., Jones A., Krakstad C., Kristensen V.N., Lambrechts D., Marchand L.L., Liang X., Lindblom A., Lissowska J., Long J., Lu L., Magliocco A.M., Martin L., McEvoy M., Meindl A., Michailidou K., Milne R.L., Mints M., Montgomery G.W., Nassir R., Olsson H., Orlow I., Otton G., Palles C., Perry J.R., Peto J., Pooler L., Prescott J., Proietto T., Rebbeck T.R., Risch H.A., Rogers P.A., Rübner M., Runnebaum I., Sacerdote C., Sarto G.E., Schumacher F., Scott R.J., Setiawan V.W., Shah M., Sheng X., Shu X.O., Southey M.C., Swerdlow A.J., Tham E., Trovik J., Turman C., Tyrer J.P., Vachon C., VanDen Berg D., Vanderstichele A., Wang Z., Webb P.M., Wentzensen N., Werner H.M., Winham S.J., Wolk A., Xia L., Xiang Y.B., Yang H.P., Yu H., Zheng W., Pharoah P.D., Dunning A.M., Kraft P., De Vivo I., Tomlinson I., Easton D.F., Spurdle A.B, & Thompson D.J. (2018). Identification of nine new susceptibility loci for endometrial cancer. Nature Communications, 9, 3166.

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MDBK Cell Culture Protocol

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Madin-Darby Bovine Kidney (MDBK, Cat.# CCL-22, ATCC, Manassas, VA) cells were seeded (1 × 10⁵ cells/well) on 24-well plates (Cat.#353047, Corning Incorporated, Corning, NY) and grown until confluency for 48 h in basal medium containing Dulbecco's Modified Eagle's Medium (Cat.#D1145, Sigma-Aldrich), 10% fetal bovine serum (Cat.#F7524, Sigma-Aldrich), 1x Penicillin-Streptomycin (Cat.#P4333, Sigma-Aldrich), and 10 mM L-glutamine (Cat.#G7513, Sigma-Aldrich). Cells were incubated at 37°C and 5% CO₂.

Felici M., Tugnoli B., Ghiselli F., Massi P., Tosi G., Fiorentini L., Piva A, & Grilli E. (2020). In vitro anticoccidial activity of thymol, carvacrol, and saponins. Poultry Science, 99(11), 5350-5355.

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Isolation and Analysis of Secretome from Adipose-Derived Mesenchymal Stem Cells

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Each donor was processed individually. fAd-MSCs (5 × 10⁵) were seeded in DMEM supplemented with 10% FBS, 2.5 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin in n T-75 flask. The culture medium was changed twice a week until reaching 80% confluence. At this time, two washes with PBS were performed, and 15 mL of DMEM without supplements (D1145, Sigma) was added to each flask. The flasks were placed in an incubator for 24 h, and then, the medium was collected and filtered using a 0.22-µm filter to isolate the secretome; the secretome was stored at −80 °C until use. The cells present at the time the medium was removed were counted using a Neubauer chamber, and their viability was evaluated with trypan blue. Subsequently, an equitable secretome mixture from each donor was lyophilized (LyoQuest, TELSTAR) and reconstituted in 500 μL of sterile bidistilled water to be analyzed in a mass spectrometer.

Villatoro A.J., Martín-Astorga M.D., Alcoholado C., Sánchez-Martín M.D, & Becerra J. (2021). Proteomic Analysis of the Secretome and Exosomes of Feline Adipose-Derived Mesenchymal Stem Cells. Animals : an Open Access Journal from MDPI, 11(2), 295.

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Isolation and Staining of Immune Cells

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All isolation solutions were at 4 °C to avoid activation of neutrophils. 4 °C phenol-red free DMEM (Sigma-Aldrich#D1145) + 0.1% sodium azide (Sigma-Aldrich #S2002-25G) + 10 mM HEPES (Gibco #15630-080) + 2% FCS (GeminiBio BenchMark #100-106) + 5 mM EDTA media was used in all cell manipulation steps. Single-cell suspensions were gently pelleted at 400 × g for 5 min at 4 °C and resuspended in 2 ml cold ACK Lysing Buffer (Lonza #10-548E) for erythrocyte lysis. After 3 min of lysis, medium was added and cells were pelleted at 400 × g for 5 min at 4 °C. Cells were then resuspended in cold media containing antibodies for staining.

Grieshaber-Bouyer R., Radtke F.A., Cunin P., Stifano G., Levescot A., Vijaykumar B., Nelson-Maney N., Blaustein R.B., Monach P.A, & Nigrovic P.A. (2021). The neutrotime transcriptional signature defines a single continuum of neutrophils across biological compartments. Nature Communications, 12, 2856.

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Cell Viability Assay in Fibroblasts

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Primary fibroblasts (1×10⁴) were plated onto 96-well plates 36 h before DMSO or TM treatment. The time points to measure the cell viability were shown in supplemental data. Following insult exposure, cells were washed twice with phosphate buffered saline (PBS) and cultured with DMEM (D1145, SIGMA) and WST-1 mixed medium for 3 hours. WST-1 was measured at an absorption of λ450 nm—λ650 nm. Data are expressed as the mean ± SEM for at least three independent experiments.

Matsuzaki S., Hiratsuka T., Taniguchi M., Shingaki K., Kubo T., Kiya K., Fujiwara T., Kanazawa S., Kanematsu R., Maeda T., Takamura H., Yamada K., Miyoshi K., Hosokawa K., Tohyama M, & Katayama T. (2015). Physiological ER Stress Mediates the Differentiation of Fibroblasts. PLoS ONE, 10(4), e0123578.

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Culturing Mouse Spermatogonial Cells

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A mouse-derived spermatogonial cell line (GC-1spg) corresponding to a spermatogonia type B stage (ATCC, CRL-2053™) was used in this work.
GC-1spg cells were cultured and maintained in Dulbecco’s Modified Eagle Medium (DMEM, D0822, Sigma-Aldrich, USA), supplemented with 10% fetal bovine serum (FBS, P211107, PAN-Biotech, Aidenbach, Germany) and 1% penicillin-streptomycin-amphotericin B solution (A5955, Sigma-Aldrich), at 37 °C in an atmosphere of 5% CO2. The culture medium was replaced every 2–3 days, and cells were sub-cultivated each time they reached a confluence of 80–90%. When performing assays, the culture medium was replaced by phenol red-free DMEM (D1145, Sigma-Aldrich) containing 10% FBS (P211107, PAN-Biotech) and adjusting the concentration of L-glutamine (3.97 mM BP379, Thermo Fisher, USA), not present in this formulation.

Duarte F., Feijó M., Luís Â., Socorro S., Maia C.J, & Correia S. (2024). Propolis Protects GC-1spg Spermatogonial Cells against Tert-Butyl Hydroperoxide-Induced Oxidative Damage. International Journal of Molecular Sciences, 25(1), 614.

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Caco-2 Cell Monolayer Permeability Assay

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Caco-2 cells were seeded onto trans-well chambers (353180, 0.4 μm pore size; Falcon) in 6-well plates and grown to confluent monolayers. DMEM supplemented with ileal content was then applied for 24 h followed by incubation for 2 h with fresh clear DMEM (D1145, Sigma) containing 10 mg/mL 4 kDa FITC-dextran (Sigma). Paracellular flux of 4 kDa FITC-dextran was assessed by taking 50 μL aliquots from the outer chamber at baseline and 100 μL aliquots from the inner chamber at 60 min. Fluorescence was measured using a spectrophotometer with excitation and emission wavelengths of 485 nm and 535 nm, respectively. Permeability coefficients (P_E) were calculated as previously described [29 (link)].

Hankir M.K., Seyfried F., Schellinger I.N., Schlegel N, & Arora T. (2021). Leaky Gut as a Potential Culprit for the Paradoxical Dysglycemic Response to Gastric Bypass-Associated Ileal Microbiota. Metabolites, 11(3), 153.

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