L2654

Manufactured by Merck Group

Sourced in United States

L2654 is a laboratory equipment product manufactured by Merck Group. It serves as a general-purpose scientific instrument used for various applications in research and analytical settings. The core function of L2654 is to provide a controlled environment for specific experimental or testing procedures. The detailed specifications and intended uses of this product are not available in this response.

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Lab products found in correlation

D1306, by Thermo Fisher Scientific (2 mentions) Anti gfap, by Agilent Technologies (2 mentions) P1585, by Merck Group (2 mentions) Kupffer cells scc119, by Merck Group (2 mentions) Bx53 fluorescence microscope, by Olympus (2 mentions) Ap20187, by Takara Bio (2 mentions) Dp80 digital camera, by Olympus (2 mentions) Mca711g, by Bio-Rad (2 mentions) In situ cell death detection kit, by Roche (2 mentions) Balb c mice, by Jackson ImmunoResearch (1 mentions) Rosettsep cocktail, by STEMCELL (1 mentions) Macrophage colony stimulating factor, by Fujifilm (1 mentions) Interleukin 4 (il 4), by BioLegend (1 mentions) Escherichia coli o26 b6, by Merck Group (1 mentions) Thp 1, by American Type Culture Collection (1 mentions) Fetal calf serum (fcs), by Merck Group (1 mentions) Streptomycin, by Merck Group (1 mentions) Penicillin, by Merck Group (1 mentions) L glutamine, by Merck Group (1 mentions) Thp 1, by Merck Group (1 mentions)

Spelling variants of this product

LPS (L2654; (4 citations) L2654-1MG (4 citations) LPS from Escherichia coli (L2654; (2 citations)

32 protocols using l2654

Evaluating Immunogenic Compounds in Mice

Cited 1 time

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Six to eight-week-old
C57BL/6, BALB/c mice and D011.10 mice were purchased from The Jackson
Laboratory. All animal experiments received prior approval by the
UC San Diego Institutional Animal Care and Use Committee (IACUC).
C57BL/6 mice (n = 5/group) were immunized in the
right gastrocnemius muscle with OVA (20 μg/animal, Worthington
Biochemical Co.) mixed with LPS (3 μg/animal, L2654, Sigma-Aldrich)
and/or compound 2D216 or 50 (100 nmol/mouse)
in a total volume of 50 μL on days 0 and 14. DMSO (10%) was
used as the vehicle. On day 17, mice were bled and OVA-specific IgG1
and IgG2c titers were measured by ELISA as previously described.^{56 (link)}

Saito T., Shukla N.M., Sato-Kaneko F., Sako Y., Hosoya T., Yao S., Lao F.S., Messer K., Pu M., Chan M., Chu P.J., Cottam H.B., Hayashi T., Carson D.A, & Corr M. (2022). Small Molecule Calcium Channel Activator Potentiates Adjuvant Activity. ACS Chemical Biology, 17(1), 217-229.

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Macrophage Differentiation and Stimulation

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Peripheral blood mononuclear cells were obtained from healthy volunteer donors who each provided written informed consent for the use of their cells in accordance with the study protocols approved by the Kumamoto University Hospital Review Board (#1169). Monocytes were isolated using RosettSep cocktail (StemCell Tech., Vancouver, Canada), plated in UpCELL culture plates (CellSeed, Tokyo, Japan), and cultured in 2% human serum, 1 ng/mL granulocyte macrophage-colony stimulating factor (WAKO, Tokyo, Japan), and 50 ng/mL macrophage-colony stimulating factor (WAKO) for 7 days to induce macrophage differentiation. Macrophages (2 × 10⁵/well) were then seeded on glass coverslips in a 12-well plate and stimulated with interleukin (IL)-10 (#093-04651, 10 ng/mL, WAKO), interferon (IFN)-α (#11200-2, 10 ng/mL, R&D Systems, Minneapolis, MN, USA), IFN-γ (#IFG4001, 10 ng/mL, WAKO), or lipopolysaccharide (LPS) (#L2654, 100 ng/mL, Sigma, St. Louis, MO, USA) for 1 day. Cells were fixed with 1% paraformaldehyde and then dried once. Cells were blocked with 1% bovine serum albumin and then stained with anti-Maf antibody. Horseradish peroxidase-labeled anti-rabbit immunoglobulin antibody (Nichirei) was used as the secondary antibody, and diaminobenzidine substrate was used for visualization.

Takeya H., Ohnishi K., Shiota T., Saito Y., Fujiwara Y., Yagi T., Kiyozumi Y., Baba Y., Yoshida N., Asano K., Tanaka M., Baba H, & Komohara Y. (2019). Maf expression in human macrophages and lymph node sinus macrophages in patients with esophageal cancer. Journal of Clinical and Experimental Hematopathology : JCEH, 59(3), 112-118.

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Cytokine-Induced Inflammatory Responses

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Cultures were treated for 24 h with 100 ng/mL of LPS from E. Coli (Sigma-Aldrich #L2654), 48 h with LPS+IFNγ (100 ng/mL LPS + 4 ng/mL IFNγ—Sigma-Aldrich #I4777), or 24 h with 20 ng/mL IL-4 (BioLegend #57430). LPC (4 ng/mL) was assayed for 24 h with quaternary cell cultures. Control cells were treated with an equivalent volume of culture medium. After treatment, conditioned media were removed and frozen. For immunocytochemistry, cells were fixed with 4% paraformaldehyde (PFA, Sigma-Aldrich #158127) in 0.1 M PBS for 20 min at RT. For ELISA, cells were removed and frozen.

Enrich-Bengoa J., Manich G., Valente T., Sanchez-Molina P., Almolda B., Solà C., Saura J., González B., Castellano B, & Perálvarez-Marín A. (2022). TRPV2: A Key Player in Myelination Disorders of the Central Nervous System. International Journal of Molecular Sciences, 23(7), 3617.

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Astrocyte Response to Inflammatory Stimuli

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To track basal and activated astrocytic response to AP, astrocytes were plated in a 48 well culture plate (10,000 cells/well) and placed into the IncuCyte S3 Live-Cell Analysis System. The IncuCyte System is a time-lapse imaging system that records cell culture changes through photographic capture of the culture well within the incubator. Cultures were acclimated to the system for a period of 6 hours, then exposed to media containing IFNy (R&D Systems, 285-IF; 200ng/ml), LPS (Sigma, L2654; 100ng/ml) or a combination of both for a period of 24 hours to induce an inflammatory response^{35 (link)}. Cells were also plated on 10-well slides and processed in tandem for immunofluorescence staining to verify activation status with anti-Gfap (DAKO, Z0334; 1:500) and counterstained with DAPI (Invitrogen, D1306; 1:1000). After activation, astrocytes were exposed to AP20187 (Clontech, 635059; 10nM or 100nM) for a period of 24 hours. The IncuCyte captured phase images of each culture well were taken every 30 minutes over this period using the following settings: (Segmentation Adjustment: 0.8, Hole Fill: 450, Adjust size (pixels): −1, Minimum area (uM^{2 (link)}): 0.1). Phase confluency difference was calculated by subtracting the final phase confluency of each image from its initial value.

Bussian T.J., Aziz A., Meyer C.F., Swenson B.L., van Deursen J.M, & Baker D.J. (2018). Clearance of senescent glial cells prevents tau-dependent pathology and cognitive decline. Nature, 562(7728), 578-582.

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Goat Model of Endotoxin-Induced Inflammation

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Sixteen goats (8 PRNP^Ter/Ter and 8 PRNP^+/+) received a dual dose of LPS (Escherichia coli O26:B6, L2654 Sigma-Aldrich, USA) intravenously with a 24-h time interval between doses; 0.1 µg/kg (day 1) and 0.05 µg/kg (day 2). The control group consisted of 10 goats (5 PRNP^Ter/Ter and 5 PRNP^+/+) and were given corresponding volumes of sterile saline. Clinical examinations, including respiratory rate, were performed at 12 time points during the first 7 h of day 1 and at 9 time points after the second LPS injection. Additionally, episodes of coughing and panting were recorded. The animals were euthanized by an overdose of pentobarbital 5 h after the second LPS challenge.

Salvesen Ø., Reiten M.R., Kamstra J.H., Bakkebø M.K., Espenes A., Tranulis M.A, & Ersdal C. (2017). Goats without Prion Protein Display Enhanced Proinflammatory Pulmonary Signaling and Extracellular Matrix Remodeling upon Systemic Lipopolysaccharide Challenge. Frontiers in Immunology, 8, 1722.

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Evaluation of Anti-inflammatory Effects of Manumycin-type Metabolites

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Human monocytic leukemia cells THP-1 (purchased from American Type Culture Collection, ATCC) were used for the evaluation of the anti-inflammatory effect of manumycin-type metabolites. THP-1 cells were cultured as described previously [24 (link)]. Briefly, THP-1 cells were cultured in RPMI-1640 medium (Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin, and streptomycin (Sigma–Aldrich, St. Louis, MO, USA) in cell culture flasks passaged every third day. To evaluate the immunomodulatory effect of streptomycetes metabolites, cells were transferred to a 12 well plate and seeded at concentration 10⁶ cells/1 mL. The total volume of cell suspension was 2 mL/well. Cells were cultured under a 5.5% CO₂ atmosphere at 37 °C. Cells were stimulated with LPS (1 μg/mL) together with manumycin-type metabolites (manumycin A, manumycin B, colabomycin E, and asukamycin) at three different concentrations (5 μM, 1 μM, and 0.25 μM). Only LPS-stimulated cells (1 μg/mL, cat. no. L-2654, Sigma Aldrich, St. Louis, MO, USA) were used as a positive control to evaluate the immunosuppressive capacity of manumycin-type metabolites. The impact of streptomycin on THP-1 viability was assessed by Trypan blue staining.

Hrdý J., Súkeníková L., Petrásková P., Novotná O., Kahoun D., Petříček M., Chroňáková A, & Petříčková K. (2020). Inhibition of Pro-Inflammatory Cytokines by Metabolites of Streptomycetes—A Potential Alternative to Current Anti-Inflammatory Drugs?. Microorganisms, 8(5), 621.

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Isolation and Culture of Peritoneal Macrophages

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Immediately after BALF was harvested, the peritoneal macrophages of the experiment mice were isolated and performed as previously described [35 (link),36 (link),38 (link),39 (link)]. Briefly, the isolated peritoneal macrophages (>95%) from each experiment mouse were adjusted to the cell density of 2 × 10⁶ cells/mL TCM medium for culture. The primary peritoneal macrophages (2 × 10⁶ cells/mL TCM medium, 0.50 mL/well) were, respectively, cultured with TCM medium (0.50 mL/well) and endotoxin lipopolysaccharide (LPS, 5.0 μg/mL, 0.50 mL/well, Sigma, L2654, St. Louis, MO, USA) in a 48-well plate for 48 h. Supernatants of cell cultures were harvested and stored at −80 °C for following pro-(IL-1β, IL-6, and TNF-α) and anti-inflammatory (IL-10) cytokine assays using a sandwich enzyme-linked immuno-absorbent assay (ELISA).

Su Y.H, & Lin J.Y. (2022). Menthone Inhalation Alleviates Local and Systemic Allergic Inflammation in Ovalbumin-Sensitized and Challenged Asthmatic Mice. International Journal of Molecular Sciences, 23(7), 4011.

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Macrophage Cytokine Response to Stimuli

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Purified alveolar macrophages (3 × 10⁵ cells/well) were stimulated with 1 µg/ml LPS (L2654, Sigma, St Louis, MO), 100 µg/ml PolyI:C (P1530, Sigma, St Louis, MO), 1 µg/ml flagellin (40067-V07E, Sino Biological Inc.) or 20 ng/ml PMA (P1585, Sigma, St Louis, MO) with a total volume of 600 µl DMEM supplemented with 10% fetal bovine serum for 48 h. Cytokines in the macrophage culture supernatants were detected. Eotaxin-2 (CCL24) (ab100681, Abcam, UK) and Arg I (KB13668, Shanghai Jiang Lai Biotechnology Co., China) were detected by ELISA. IL-6 (558301), IL-1β (560232), IL-10 (558300) and IL-13 (558349) were detected by Flow CBA Kit (BD Pharmingen, San Diego, CA, USA).

Cheng M., Chen Y., Wang L., Chen W., Yang L., Shen G., Xu T., Shen G., Tian Z, & Hu S. (2017). Commensal microbiota maintains alveolar macrophages with a low level of CCL24 production to generate anti-metastatic tumor activity. Scientific Reports, 7, 7471.

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Endothelial Activation by TNF-α and LPS

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Endothelial activation was induced by exposing the cells to various concentrations of TNF-α (50103 ab9642 Active TNF alpha full length protein, Abcam Ltd.) for 24 h. TNF-α was diluted to a final stock concentration of 200 μg ml⁻¹ in dd.H₂O with 0.1% BSA and filtered through a 0.22-μm filter. IκBα is an inhibitor of the NFκB complex and was used to prevent TNF-α induced inflammation^{50 (link)}. IκBα (SRP5195-50UG, Sigma Aldrich Ireland) and was diluted in dd.H₂O with 0.1% BSA and filtered through a 0.22-μm filter to a final stock concentration of 200 μg ml⁻¹. A second method of endothelial activation was implemented by exposing the cells to various concentrations of LPS (lipopolysaccharide, L2654, Sigma Aldrich, Ireland) for 24 h. The LPS solution was diluted in dd.H₂O with 0.1% BSA and filtered through a 0.22-μm filter to a final stock concentration of 1 mg ml⁻¹.

King D., Glynn M., Cindric S., Kernan D., O’Connell T., Hakimjavadi R., Kearney S., Ackermann T., Berbel X.M., Llobera A., Simonsen U., Laursen B.E., Redmond E.M., Cahill P.A, & Ducrée J. (2019). Label-Free Multi Parameter Optical Interrogation of Endothelial Activation in Single Cells using a Lab on a Disc Platform. Scientific Reports, 9, 4157.

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Stimulation of Isolated Immune Cells

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Isolated cells from 3 individual donors were stimulated with a pan-bacterial cocktail comprised of lipopolysaccharide from Escherichia coli O26:B6 (LPS) (Sigma, L2654) at 1 µg/mL and heat-killed Staphylococcus aureus (HKSA) (InvivoGen, tlrl-hksa) at 10⁸ cells/mL. Commercial stocks were reconstituted at 100 × in UltraPure™ DNase/RNase-Free Distilled Water (Invitrogen, 10977015) and stored as individual use aliquots at − 20 °C. Unstimulated cells received 2 µL water as vehicle. Cells were incubated for 24 h in RPMI containing 1% human serum plus the relevant stimulus at 37 °C with gentle agitation at 80 rpm on an orbital shaker (Eppendorf, M1190-0000) housed inside a standard tissue culture incubator (5% CO₂, humidified). At endpoint, cells were collected by centrifugation at 400 g for 5 min at room temperature and supernatants were collected, clarified at > 16000 g for 5 min, and stored at − 20 °C.

Johnson R.K., Overlee B.L., Sagen J.A, & Howe C.L. (2022). Peripheral blood mononuclear cell phenotype and function are maintained after overnight shipping of whole blood. Scientific Reports, 12, 19920.

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