FLS-RA (6 ∗ 105 cells/ml) were seeded into the 96-well plates, and all cells were pretreated with 10 ng/ml IL-1 for 8 h. When screening the suitable berberine (Shifeng, C20H18NO4) content, cells were grown in the mediums supplemented with various berberine doses (0, 6.25, 12.5, 25, 50, and 100 μM) for 12 h, 24 h, and 48 h, respectively. When screening the appropriate LPA (Sigma, L7260) content, cells were planted in the medium containing various LPA doses (0, 5, and 10 μM) for 24 h, respectively. When selecting the suitable Ki16425 (Cayman, 10012659) content, cells were plated with various Ki16425 doses (0, 10, and 20 μM) for 24 h, respectively. Afterwards, the cells were incubated with berberine (12.5 and 25 μM) for 1 h and then treated with 10 μM LPA or Ki16425 for 24 h, so as to investigate the mechanism of berberine in inhibiting cell proliferation. Subsequently, the wells were incubated with 1 mg/ml MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma, m2128) at 37°C for 4 h, and then 100 μL dimethyl sulfoxide was added into each well. After the purple formazan crystals were completely dissolved, the optical density (OD) was measured at the wavelength of 490 nm.
L7260
Manufactured by Merck Group
Sourced in United States
The L7260 is a laboratory equipment product from the Merck Group. It is designed to perform core functions required in laboratory settings. A detailed description is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Neg030x, by PerkinElmer (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Ki16425, by Cayman Chemical (1 mentions)
M2128, by Merck Group (1 mentions)
Lsm 900, by Zeiss (1 mentions)
Ab112129, by Abcam (1 mentions)
S2767, by Selleck Chemicals (1 mentions)
S1105, by Selleck Chemicals (1 mentions)
Ki16425, by Selleck Chemicals (1 mentions)
Ly294002, by Selleck Chemicals (1 mentions)
B0560, by Merck Group (1 mentions)
I0634, by Merck Group (1 mentions)
C9754, by Merck Group (1 mentions)
Ct04 a, by Cytoskeleton (1 mentions)
Sml0341, by Merck Group (1 mentions)
Y0503, by Merck Group (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Mcf 7 breast, by American Type Culture Collection (1 mentions)
Plan apochromat 63 1.40 oil ph3 lens, by Zeiss (1 mentions)
A0082, by Agilent Technologies (1 mentions)
10 protocols using l7260
1
Berberine Inhibits Cell Proliferation
2
Gabapentin Modulates Calcium Dynamics
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Gabapentin (100 μM) pretreated primary motor neurons for 36h, and calcium ion indicator (Abcam, Cat# ab112129) would show the changes of Ca2+ in neurons after axon injury caused by LPA (Sigma-Aldrich, Cat# L7260) in the form of fluorescence. After the addition of LPA (2 μM), fluorescence imaging of living cells was performed immediately, generating an image every 30s for 100 cycles under a confocal microscope (Zeiss, LSM900).
3
Regulation of Granulosa-like Tumor Cells
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Human granulosa-like tumor cell line (KGNs, iCell-h298, icell bioscience Inc, Shanghai, China) were cultured in RPMI 1640 containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C with 5% CO2. KGNs were treated with various concentrations of LPA (0, 5, 10, 20 and 40 μM; L-7260, Sigma-Aldrich, Shanghai, China) for 24 h when performing cell counting kit-8. Then, KGNs were treated with 20 μM LY294002 (an inhibitor of PI3K; S1105, Selleck, Shanghai, China), as well as 5 mM 3 methyladenine (3MA, autophagy inhibitor; S2767, Selleck) for 24 h, before exposure to 10 μM LPA for 24 h. LPAR1 inhibitor and controls were purchased from the GenePharm (Shanghai, China), and KGNs were treated with 10 μM Ki16425 (LPAR1 and LPAR3 antagonist; S1315, Selleck) for 24 h, before exposure to 10 μM LPA for 24 h.
4
Binding Assay for LPA1 Receptor Activation
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Example 1
Binding test of GTPγS to LPA1 receptor
5 μg of the membrane fraction of RH 7777 cells expressed with human LPA1 receptor (A324, ChanTest) was suspended in a reaction buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 10 μM GDP, 5 μg saponin, 0.2% BSA, 0.1 nM [35S]GTPγS (NEG030X, Perkin Elmer), pH 7.4), and the test compound dissolved in DMSO in various concentrations was added. After preincubation at 30° C. for 15 minutes, LPA (L7260, Sigma, the final concentration of 100 nM) was added, and the suspension was incubated at 30° C. for 30 minutes. The membrane fraction was collected onto a glass fiber filter (GF/B, Whatman) by using a cell harvester (M30, Brandel), and washed with a 10 mM phosphate buffer (pH 7.4). The radioactivity of the membrane fraction was measured using a liquid scintillation analyzer (2900TR, Packard) and the concentration of the test compound which inhibits the binding of LPA1 receptor and [35S]GTPγS by 50%, (IC50) was determined by non-linear regression analysis using EXSAS (version 7.6.0, Arm Systex).
The compound of the present invention showed the excellent activity in this test, and, for example, the IC50 values of Compound Nos. I-11, I-24, I-28, I-38, I-42, I-51, I-52, I-56, I-60, I-76, I-100, I-104, I-112, I-122, I-126, I-140, and I-152 were 200 nM or less.
5
Lipid Signaling Pathway Modulation
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LPA (18:1, L7260), phosphatidylserine (PS, P7769-5MG) and phosphatidylcholine (PC, P3556-25MG) were purchased from Sigma and all other lipid chemicals were purchased from Avanti Polar Lipids. Other Lipids shown in Fig. 1D and Fig. S1D are lyso phosphatidylserine (LPS, 860081), lyso-phosphatidylcholine (LPC, 850092P), phosphatidylethanolamine (PE, 840465P), lyso phosphatidylethanolamine (LPE, 858127c), phosphatidylglycerol (PG), lyso phosphatidylglycerol (LPG, 830071), phosphatidic acid (PA, 840861P), sphingomyelin (SPH, 860062P), phosphatidylinositol (PI, 850142P), phosphatidylinositol 3-phosphate (PI3P, 850150P), phosphatidylinositol(3,4)-bisphosphate (PI3,4P, 850153P), cardiolipin (CL, 710335P), 16:0 LPA (857123P), 18:0 LPA (857125c) and 20:4 LPA (857128p).
The LPAR1/3 inhibitor Ki16425 (40 μM, S1315, Selleck), HDAC6 inhibitor tubacin (2 μM, HY-13428, MCE), CaM inhibitor CMZ (5 μM, 2561, R&D), calcium chelator EGTA (0.5 mM, HY-D0973, MCE) or DMSO vehicle were added to RPE-1 cells 30 min prior to the treatment of serum or LPA.
The LPAR1/3 inhibitor Ki16425 (40 μM, S1315, Selleck), HDAC6 inhibitor tubacin (2 μM, HY-13428, MCE), CaM inhibitor CMZ (5 μM, 2561, R&D), calcium chelator EGTA (0.5 mM, HY-D0973, MCE) or DMSO vehicle were added to RPE-1 cells 30 min prior to the treatment of serum or LPA.
6
Establishing and Treating Cell Lines
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Human HT-1080 fibrosarcoma, A431 epidermoid carcinoma, MCF-7 breast cancer, and human embryonic kidney (HEK) 293T cells were obtained from the American Type Culture Collection and cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing glucose (4.5 g/liter), l -glutamine, and sodium pyruvate (11995073, Gibco). Medium was supplemented with 10% heat-inactivated FBS (16140071, Gibco) and 1% penicillin-streptomycin (10,000 U/ml; 15140122, Gibco). Cells were maintained at 37°C with 5% CO2. A431 cells expressing GFP-E-cad were a gift from V. Bruntons (EH4 2XR, University of Edinburgh, UK) (63 (link)). In select experiments, cells were treated with the following pharmacological agents: Y-27632 (20 μM; Y0503, Sigma-Aldrich), ionomycin (0.5 and 1 μM; I0634, Sigma-Aldrich), blebbistatin (50 μM; B0560, Sigma-Aldrich), Rho inhibitor I (1 μg/ml; CT04-A, Cytoskeleton Inc.), LPA (50 μM; L7260, Sigma-Aldrich), paclitaxel (taxol equivalent) (1 μM; P3456, Thermo Fisher Scientific), colchicine (125 μM; C9754, Sigma-Aldrich), and importazole (50 μM; SML0341, Sigma-Aldrich).
7
Binding Assay for LPA1 Receptor Activation
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Example 1
Binding test of GTPγS to LPA1 receptor
5 μg of the membrane fraction of RH 7777 cells expressed with human LPA1 receptor (A324, ChanTest) was suspended in a reaction buffer (20 mM HEPES, 100 mM NaCl, 10 mM MgCl2, 10 μM GDP, 5 μg saponin, 0.2% BSA, 0.1 nM [35S]GTPγS (NEG030X, Perkin Elmer), pH 7.4), and the test compound dissolved in DMSO in various concentrations was added. After preincubation at 30° C. for 15 minutes, LPA (L7260, Sigma, the final concentration of 100 nM) was added, and the suspension was incubated at 30° C. for 30 minutes. The membrane fraction was collected onto a glass fiber filter (GF/B, Whatman) by using a cell harvester (M30, Brandel), and washed with a 10 mM phosphate buffer (pH 7.4). The radioactivity of the membrane fraction was measured using a liquid scintillation analyzer (2900TR, Packard) and the concentration of the test compound which inhibits the binding of LPA1 receptor and [35S]GTPγS by 50%, (1050) was determined by non-linear regression analysis using EXSAS (version 7.6.0, Arm Systex).
The compound of the present invention showed the excellent activity in this test, and, for example, the IC50 values of Compound Nos. I-11, I-24, I-28, I-38, I-42, I-51, I-52, I-56, I-60, I-76, I-100, I-104, I-112, I-122, I-126, I-140, and I-152 were 200 nM or less.
8
Cell Pretreatment for Nucleus Imaging
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Prior to NE wrinkling analysis, cells were treated with 25 µM ML7 (ab120848, Abcam, Waltham, MA, USA) for 1 h before fixing, or with 50 µM LPA (L7260, Sigma-Aldrich) for 2 h post-seeding, followed by a wash with growth medium before incubating cells in fresh growth medium until the fixation time-point. Prior to Ki67 analysis, treatment with 10 µM ML7 or LPA was conducted overnight. In all of these conditions, the cells were fixed at 45 h post-seeding.
9
Endothelial Injury Repair via Monocytes
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Endothelial injury was induced in murine aortas, longitudinally open, through the exposure to Lipopolyssacharides (LPS) (0.5 µg/mL; L2880-Sigma-Aldrich) plus Lysophosphatidic acid (LPA) (0.5 µg/mL; L7260-Sigma-Aldrich), over 24 h. Afterwards, aortas were rinsed with 1× PBS and embedded in Matrigel with 50 ng/mL VEGF plus 10 U/mL heparin with human Day 3 monocytes, cultured in EBM2 supplemented with 50 ng/mL VEGF plus 10 U/mL heparin, for 5 days. Aortas were removed from Matrigel and fixed with 4% paraformaldehyde (PFA) for 2 h, and rinsed with blocking buffer (1× PBS + 10% horse serum + 0.3% Triton X-100). The aortas were incubated with primary antibody diluted in 1% BSA-PBS (v/w) (vWF—1:200, A0082; Dako), overnight at 4 °C, followed by two times rinsing with blocking buffer and a 2 h incubation with secondary antibody (1:500 in 1% BSA-PBS (v/w); Alexa Fluor® 594 goat anti-rabbit) at room temperature. After the final washes with blocking buffer, aortas were mounted in slides with in VECTASHIELD media with DAPI. Images were acquired using an LSM 710 (Zeiss) confocal microscope equipped with a Plan-Apochromat 63/1.40 Oil Ph3 lens and Zen Blue 2010b SP1software (2010b; Zeiss).
10
Modulating Rho Activity in Cell Culture
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To inhibit Rho activity, C3 (Cytoskeleton; CT04-A) was applied at 0.3 mg/ml while Rho activation was achieved via oleoyl-l -α-lysophosphatidic acid sodium salt (LPA; Sigma L7260) applied at 50 mM (for monolayer experiments) or 20 mM (for PA gel studies). Small molecules were added 2 h postseeding and replaced every day of culture with complete media changes.
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