M4655

Manufactured by Merck Group

Sourced in United States

The M4655 is a laboratory equipment product from Merck Group. It is designed for general laboratory use. The core function of this product is to provide a reliable and consistent platform for various laboratory procedures and experiments. No further details on the intended use or specific applications are provided.

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Lab products found in correlation

17 protocols using m4655

Osteoblast Differentiation from Mouse Bone Marrow

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Five normal mice in the control group were anesthetized and euthanized, the femur and tibia were separated, the bone marrow was washed with pre‐cooled PBS (79378, Sigma) containing 2% FBS and placed in serum‐free ɑ‐MEM medium (M4655, Sigma) for later use. The cell suspension was filtered through a 100 mesh sieve, centrifuged at 250 g for 5 min, and the supernatant was discarded; 10 times the cell volume of red blood cell lysis buffer (R7757, Sigma) was added and centrifuged at 250 g for 5 min, and the supernatant was discarded. Bone marrow cells were suspended in ɑ‐MEM medium containing 10% FBS and incubated at 37°C in the incubator of 5% CO₂ for 24 h. The supernatant was collected; the supernatant was discarded after centrifugation at 250 g for 5 min and then incubated in ɑ‐MEM medium containing 25 ng/mL M‐CSF (SRP3221, Sigma) and 10% FBS for 3 days. The adherent cells were used as osteoblastic progenitor cells. Finally, ɑ‐MEM medium containing 25 ng/mL M‐CSF, 50 ng/mL RANKL (R0525, Sigma), and 10% FBS was used to incubate for 6 days to induce osteoblast differentiation. During the induction of differentiation, 5 ng/mL TGF‐β1 was added or not as TGF‐β1+ group or TGF‐β1− group to observe the effect of TGF‐β1 on osteoclast differentiation.
²⁹ (link)

Wang K., Wu Z., Gong C., Zhao G, & Zhang H. (2023). TGF‐β1 Inhibits Osteoclast Differentiation and Abnormal Angiogenesis in Intervertebral Disc Degeneration: Evidence from RNA Sequencing and Animal Studies. Orthopaedic Surgery, 16(1), 167-182.

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Culturing Human Neuroblastoma Cell Lines

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IMR-32 human neuroblastoma cell line was cultured in EMEM medium (M4655, Sigma-Aldrich) supplemented with 10% fetal calf serum (10270106, Gibco), 1% non-essential amino acid solution (M7145, Sigma-Aldrich), 1 mM sodium pyruvate (S8636, Sigma-Aldrich) and 50 µg/ml gentamicin (G1397, Sigma-Aldrich). CHP-134 cells were grown in RPMI 1640 medium (R8758, Sigma-Aldrich) supplemented with 10% FCS and 50 µg/ml gentamicin. LAN-1 cells were cultured in EMEM/F-12 (N6658, Sigma-Aldrich) medium diluted in 1:1 ratio, supplemented with 10% FCS, 1% non-essential amino acid solution, 1 mM sodium pyruvate and 50 µg/ml gentamicin, while LAN-5 cells in RPMI 1640 medium supplemented with 20% FCS and 50 µg/ml gentamicin. For preparation of positive controls, IMR-32 and CHP-134 cells were cultured in amino acids-deprived Earle’s Balanced Salt (E2888, Sigma-Aldrich), supplemented with 10% FCS for 24 h. All cell lines were grown at 37 °C in a 5% CO₂ atmosphere.

Durbas M., Pabisz P., Wawak K., Wiśniewska A., Boratyn E., Nowak I., Horwacik I., Woźnicka O, & Rokita H. (2018). GD2 ganglioside-binding antibody 14G2a and specific aurora A kinase inhibitor MK-5108 induce autophagy in IMR-32 neuroblastoma cells. Apoptosis, 23(9), 492-511.

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Spatially-Targeted Cre Recombination in Mice

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Mice lacking Cre expression were injected with an adenovirus expressing Cre, Ad5CMVCre (University of Iowa Viral Vector Core, VVC-U of Iowa-5), to activate recombination via Cre recombinase. Virus was prepared by mixing 25 μL of Ad-Cre with 600 μL minimal essential medium (Sigma-Aldrich, M4655). 3 μL of 2 M CaCl₂ was added to each virus preparation, mixed, and incubated for 15 min at room temperature before injection of 50 μL into the gastrocnemius muscle. To activate CreER in a spatially restricted manner, a 5 mg/mL 4-hydroxytamoxifen (4-OHT, Sigma, H7904) solution in 25% ethanol/75% corn oil was administered via intramuscular injection. The solution was prepared by dissolving 4-OHT in ethanol and incubating at 60°C for 2 hours with shaking. Next, corn oil was added at a 3 : 1 corn oil to ethanol ratio, with continuous shaking at 65°C for 1 hour. Aliquots were stored at −80°C. 20 μL was injected into the gastrocnemius of experimental mice. All mice were anaesthetized with 2% isoflurane prior to any injection or procedure. All animal studies were performed in accordance with protocols approved by the Duke University Institutional Animal Care and Use Committee.

Chen M., Xu E.S., Leisenring N.H., Cardona D.M., Luo L., Ma Y., Ventura A, & Kirsch D.G. (2019). The Fusion Oncogene FUS-CHOP Drives Sarcomagenesis of High-Grade Spindle Cell Sarcomas in Mice. Sarcoma, 2019, 1340261.

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Aspergillus fumigatus Secretome under Oxygen Conditions

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A. fumigatus ATCC 13073 strain was obtained from the American Type Culture Collection. It was grown for five days on Sabouraud dextrose agar (SDA) (Oxoid, Basingstoke, United Kingdom) at 30°C. After a five-day period of incubation, A. fumigatus conidia were collected and resuspended in minimal essential medium (MEM) (Sigma Chemical Co., M4655, St. Louis, MO) at the concentration 1x10⁶ CFU/mL. MEM was used instead of complex and rich media, because it facilitates the detection of secreted products. Media with fetal calf serum (FCS) can mask small secreted molecules and make their detection impossible. Sixty milliliters of conidial suspension were put into 250-ml flasks, incubated under oxygen (20% O₂) and non-oxygen (0% O₂) conditions at 37°C with agitation at 140 rpm. Six different A. fumigatus CF samples were collected by centrifugation: (i) under aerated conditions on days 1, 3 and 6 (A. fumigatus CF AE-1; A. fumigatus CF AE-3; A. fumigatus CF AE-6, respectively) and (ii) under non- aerated conditions on days 1, 3 and 6 (A. fumigatus CF AN-1; A. fumigatus CF AN-3; A. fumigatus CF AN-6, respectively). Supernatants were filtered through a 0,22 μm Millipore filters and stored at -20°C until use.

Arsic Arsenijevic V.S., Pekmezovic M.G., Rajkovic K.M., Vekic B.P., Barac A.M., Tasic-Otasevic S, & Petkovic L.D. (2014). In vitro Protease Inhibition and Cytotoxicity of Aspergillus fumigatus Biomolecules Secreted under Long-Term Aerated Conditions. International Journal of Medical Sciences, 11(11), 1133-1139.

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Culturing Human Glioblastoma Cells

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A human brain glioblastoma epithelial cell line (U87) was purchased from the Korean Cell Line Bank (Seoul, Republic of Korea) and cultured in minimum essential Eagle’s medium (M4655; Sigma Gangnam-gu, Republic of Korea) in a humidified atmosphere (5% CO2) at 37 °C. All media were supplemented with 10% fetal bovine serum (FBS; Corning, Glendale, CA, USA), 100 units/mL penicillin, and 100 units/mL streptomycin. Cells (1 × 104 cells/well) were seeded in 96-well plates and incubated at 37 °C with 5% CO2 for 24 h.

Oh E.J., Jeon J.S., Wang Q.W, & Kim J.K. (2023). Early immune response of neuronal cells (U87) to heavy metal Cd or Pb exposure. Environmental Analysis, Health and Toxicology, 38(1), e2023004.

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Lung Cancer Cell Line Cultivation

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Human LUAD (HCC827), SCC (H520, H446, and SK-MES-1), and LCC (H1299) cell lines were purchased from the American Type Culture Collection. Human SCLC cell lines (LK-2 and SBC-3) were purchased from the Japanese Collection of Research Bioresources Cell Bank and validated by short tandem repeat profiling (National Institute of Biomedical Innovation). All cell lines were routinely tested for Mycoplasma and confirmed to have no contamination using a LookOut Kit (MP0035; Sigma–Aldrich) according to the manufacturer’s instructions. H1299, HCC827, H520, H446, and LK-2 were maintained in RPMI1640 (187-02705 for H520 and H446, FUJIFILM Wako; R8758 for LK-2, Sigma–Aldrich). SK-MES-1 and SBC-3 were maintained in Eagle’s minimal essential medium (M4655; Sigma–Aldrich). All media contained 10% fetal bovine serum (10270106; Sigma–Aldrich) and 100 U/ml penicillin and 10 μg/ml streptomycin (P4333; Sigma–Aldrich). Cells were cultured at 37 °C in a 5% CO₂ atmosphere.

Kondo K., Harada Y., Nakano M., Suzuki T., Fukushige T., Hanzawa K., Yagi H., Takagi K., Mizuno K., Miyamoto Y., Taniguchi N., Kato K., Kanekura T., Dohmae N., Machida K., Maruyama I, & Inoue H. (2022). Identification of distinct N-glycosylation patterns on extracellular vesicles from small-cell and non–small-cell lung cancer cells. The Journal of Biological Chemistry, 298(6), 101950.

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Quantifying Senescent Fibroblast Dynamics

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The human diploid fibroblast WI-38 cell line (American Type Culture Collection, ATCC® CCL75™) was cultured in Minimum Essential Medium Eagle (Sigma-Aldrich, M4655) containing 10% FBS (Sigma-Aldrich, F2442), 1× Non-Essential Amino Acids Solution (Gibco, 11140050), 1 : 100 L-Glutamine–Penicillin–Streptomycin solution (Sigma-Aldrich, G6784), and maintained in a 37°C, 5% CO2 incubator. WI-38 fibroblasts were cultured from a cumulative population doubling (CPD) level of 37 representing a proliferative state based on Sidler et al. [20 (link), 21 (link)], to a senescent state defined by a failure to double after one week (CPD 56-60) [6 (link)]. Serial culture of WI-38 was performed by seeding cells at a density of 7000 cells/cm² and trypsinization with 0.25% (weight/volume) Trypsin-0.53 mM EDTA solution every 3-4 days until senescent status was reached. Cell numbers were recorded for estimated CPD and doubling time. Doubling time was calculated as follows: Doubling time = duration∗log₂ (2)/log₂ (Final Cell Concentration)–log₂ (Initial Cell Concentration) [22 ].

Chen Y.H., Zhang X., Ko K.Y., Hsueh M.F, & Kraus V.B. (2022). CBX4 Regulates Replicative Senescence of WI-38 Fibroblasts. Oxidative Medicine and Cellular Longevity, 2022, 5503575.

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Swine Influenza Virus Propagation

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Madin-Darby canine kidney (MDCK) (ATCC, #CRL-2936) cells were maintained in Minimal Essential Medium (MEM) (Sigma-Aldrich, M4655, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Ottawa, ON, Canada 16000-044), and were kept in a humidified 5% CO₂ incubator at 37 °C. A/Swine/Alberta/SD0435/2019 (H3N2) [SD435] and A/Swine/Manitoba/SD0467/2019 (H1N2) [SD467] swIAV were isolated from field samples at the Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, SK, Canada. The vaccine viruses SD191−R342V and SD69-K345V were rescued as previously described [14 (link)]. All viruses were grown in MDCK cells in the presence of 0.2% bovine serum albumin (BSA) (Sigma-Aldrich, A7030) with either 1 μg/mL L-[(toluene-4-sulphonamido)-2-phenyl] ethyl chloromethyl ketone (TPCK)-trypsin (WT viruses) or 0.5 μg/mL human neutrophil elastase (elastase-dependent viruses) (Sigma-Aldrich, E8140).

Aubrey L., Barron-Castillo U., Detmer S, & Zhou Y. (2022). A Bivalent Live Attenuated Influenza Virus Vaccine Protects against Drifted H1N2 and H3N2 Clinical Isolates in Swine. Viruses, 15(1), 46.

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Culturing HEK Cells in Optimized Media

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All the cells were cultured using Minimal Essential Media (M4655, sigma) with 1g/L of glucose supplemented with 10% Fetal Bovine Serum, 1mM Sodium Pyruvate (Gibco) and 1% Penicillin and streptomycin (Gibco), incubated at 37°C at 5% CO2 incubator. HEK (Flp-InTM T-RExTM 293) cells were obtained from Björn Stork from Institute of Molecular Medicine I, Düsseldorf, Germany.

Anand R., Strecker V., Urbach J., Wittig I, & Reichert A.S. (2016). Mic13 Is Essential for Formation of Crista Junctions in Mammalian Cells. PLoS ONE, 11(8), e0160258.

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Differentiation and Stimulation of BMDCs and BMDMs

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B16-F1 (Riken BRC, Ibaraki, Japan) cells were grown in D-MEM (High Glucose, 043-30085, FUJIFILM Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS). RAW 264.7 (Riken BRC) cells were grown in MEM (Minimum Essential Medium Eagle, M4655, Sigma-Aldrich, St. Louis, MO, USA) containing 10% FBS. BMDCs were generated as described previously (Kanemaru et al., 2017 (link)). Briefly, mouse bone marrow cells were obtained from femurs and differentiated into DCs in MEM with 10% FBS containing 10 ng/ml granulocyte macrophage colony-stimulating factor (G M-CSF; Prospec, East Brunswick, NJ, USA). Medium was replaced with fresh medium containing G M-CSF every two days. On day 6, cells were collected. BMDMs were also generated as described previously (Kanemaru et al., 2017 (link)). Bone marrow cells were obtained and incubated in MEM with 10% FBS containing 3 ng/ml macrophage colony-stimulating factor (M-CSF, PeproTech, Rocky Hill, NJ, USA). Medium was changed on day 3. On day 5, macrophages were harvested. BMDCs and BMDMs were stimulated with LPS (100 ng/ml; LPS-SM ultrapure, InvivoGen, San Diego, CA, USA) and/or L-(+)-Lactic acid (Sigma-Aldrich), and/or sodium L-lactate (Sigma-Aldrich).

Kanemaru H., Mizukami Y., Kaneko A., Tagawa H., Kimura T., Kuriyama H., Sawamura S., Kajihara I., Makino K., Miyashita A., Aoi J., Makino T., Masuguchi S., Fukushima S, & Ihn H. (2021). A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells. iScience, 24(9), 103067.

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