PDLF of 3rd to 5th passage were used for the experiments. A total of 70,000 PDLF in 2 ml DMEM per well were randomly seeded onto 6-well-plates, and preincubated for 24 h with or without addition of 100 μM of histamine (H7125, Sigma Aldrich, Munich, Germany) (50, 100 and 200 μM in receptor expression experiments). After that time PDLF were left untreated or a glass plate (2g/cm2) was applied for another 48 h to simulate orthodontic pressure in the periodontal ligament according to an established and published in-vitro model [30 (link),31 (link)] (Fig 1 ). To test for histamine receptor (HR) interaction, we additionally incubated PDLF with 100 μM of H1R antagonist cetirizine (C3618, Sigma Aldrich, Munich, Germany), H2R antagonist ranitidine (R101, Sigma Aldrich, Munich, Germany) or H4R antagonist JNJ7777120 (J3770, Sigma Aldrich, Munich, Germany), respectively, two hours prior to histamine application. The used antagonist concentrations were adopted from the concentration of a H1R antagonist previously used and published in experiments on nasal fibroblasts [32 (link)]. Then PDLF were preincubated for 24 h followed by pressure application for another 48 h as described above. We then analyzed gene expression (RT-qPCR) and protein expression (Western Blot, ELISA).
H7125
Manufactured by Merck Group
Sourced in United States
The H7125 is a laboratory equipment product manufactured by Merck Group. It is designed for specific laboratory applications, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. More information may be available from Merck Group's technical documentation.
Automatically generated - may contain errors
Lab products found in correlation
M2028, by Merck Group (2 mentions)
Ranitidine, by Merck Group (1 mentions)
Histamine, by Merck Group (1 mentions)
Jnj7777120, by Merck Group (1 mentions)
Formamide, by Merck Group (1 mentions)
E2129, by Merck Group (1 mentions)
Multiskan go spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Imager two microscope, by Zeiss (1 mentions)
Pluronic acid, by Thermo Fisher Scientific (1 mentions)
X well, by Sarstedt (1 mentions)
Par2 ap, by Merck Group (1 mentions)
Fura 2 am, by Molecular Devices (1 mentions)
F6886, by Merck Group (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
I0634, by Merck Group (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Capsaicin, by Merck Group (1 mentions)
D7139, by Thermo Fisher Scientific (1 mentions)
D1817, by Thermo Fisher Scientific (1 mentions)
9 protocols using h7125
1
Histamine Modulation of Periodontal Fibroblasts
2
Histamine-Induced Vascular Permeability
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Adult mice (20 g body weight) received histamine (2.5 mg; Sigma, H7125) in phosphate-buffered saline (PBS) via intravenous (i.v.) injection. Hearts were harvested 36 h later and digested by the Langendorff method.
For the Evans blue (EB) dye assay, 4 h after histamine injection mice received 100 μl of 0.5% EB (Sigma, E2129) in PBS via i.v. injection. Mice were euthanized 30 min later and hearts were perfused with 10 ml of PBS through the left ventricle to remove excess EB from vessels. Hearts were then desiccated (24 h, 60 °C), followed by EB dye elution in formamide (Millipore, 109684; 24 h, 65 °C). To analyze the amount of EB in the supernatant, samples were placed in a 96-well plate and read with a Multiskan GO Spectrophotometer (Thermo Scientific) at 620 and 720 nm, correcting for contaminating heme pigments: A620 (EB) = A620-(1.426 × A720+ 0.030) [37 ]. The EB concentration was calculated against a standard curve.
For the Evans blue (EB) dye assay, 4 h after histamine injection mice received 100 μl of 0.5% EB (Sigma, E2129) in PBS via i.v. injection. Mice were euthanized 30 min later and hearts were perfused with 10 ml of PBS through the left ventricle to remove excess EB from vessels. Hearts were then desiccated (24 h, 60 °C), followed by EB dye elution in formamide (Millipore, 109684; 24 h, 65 °C). To analyze the amount of EB in the supernatant, samples were placed in a 96-well plate and read with a Multiskan GO Spectrophotometer (Thermo Scientific) at 620 and 720 nm, correcting for contaminating heme pigments: A620 (EB) = A620-(1.426 × A720+ 0.030) [37 ]. The EB concentration was calculated against a standard curve.
3
Imaging of C. elegans Mating and Restrained Behavior
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Imaging was performed in an upright Zeiss Axio Imager two microscope with a 470 nm LED and a GYR LED (CoolLED) with a dual-band excitation filter F59-019 and dichroic F58-019 (Chroma) in the microscope turret. Emission filters ET515/30M and ET641/75 and dichroic T565lprx-UF2 were placed in the cube of a Cairn OptoSplit II attached between the microscope and an ORCA-Flash four camera (Hamamatsu). Acquisition was performed at 20 fps. Imaging during mating was performed with a 20x long working distance objective (LD Plan-NEOFLUAR numerical aperture 0.4), placing the male on an agar pad with food and 20 hermaphrodites. The ~50 mm per side agar pad was cut out from a regular, seeded NGM plate and placed on a glass slide. The hermaphrodites were placed in a ~ 100 mm2 centre region. A fresh pad was used every two recordings.
Imaging in restrained animals was performed for 2.5–3 min with a 63x objective (LD C- apochromat numerical aperture 1.15). Animals were glued with Wormglu along the body to a 5% agarose pad on a glass slide and covered with M9 or 20 mM histamine (Sigma, H7125) and a coverslip.
Imaging in restrained animals was performed for 2.5–3 min with a 63x objective (LD C- apochromat numerical aperture 1.15). Animals were glued with Wormglu along the body to a 5% agarose pad on a glass slide and covered with M9 or 20 mM histamine (Sigma, H7125) and a coverslip.
4
Murine Colonic Explant Culture for NLRP6 and PPAR-γ
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An ex vivo system of cultured mouse colonic explants was constructed according to the methods in a previous report [13 (link)] (Additional file 2 ). Colons from eight-week-old normal C57BL/6N mice were longitudinally opened and washed thoroughly by flushing several times with cold HBSS containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 50 μg/mL metronidazole. The colon tissues were cut into approximately 4-mm2 fragments, placed on presoaked Gelfoam rafts, and cultured in 1 mL of RPMI 1640 medium containing 0.01% BSA, 200 U/mL penicillin, 200 μg/mL streptomycin, and 1% Fungizone. The colonic explants were incubated in the presence of a mixture (3:1:1 ratio) of sodium acetate, sodium propionate, and sodium butyrate with histamine (H7125; Sigma-Aldrich), pioglitazone, or GW9662 (a PPAR-γ antagonist) for 24 h according to the experimental design. The culture medium and the colonic explants were collected to test the activation of the NLRP6 inflammasome and the transcription of PPAR-γ.
5
Characterizing Calcium Signaling in Differentiated Stem Cells
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The differentiated SKPs (up to > 15 days with maturation medium) were seeded on X-well (Sarstedt) loaded with 4 µM Fura 2-AM (Molecular Devices, Sunnyvale, CA, USA) and 2 µM pluronic acid (Thermo Scientific, Waltham, MA, USA) in the dark for 45 min at 37 °C and 5% CO2. The cells were washed to remove the excess dye, and intracellular Ca2+ measurements were performed according to the protocol described by Sakka et al. [28 (link)]. For the data analysis, the F340/F380 ratio of emitted fluorescence signals intensity recorded at 340 and 380 nM excitation wavelengths was calculated for each measurement time point. The amplitude of the elicited Ca2+ responses was measured by calculating the difference between the basal and maximal F340/F380 ratio values. Cells were stimulated with capsaicin at 10 µM (Sigma, M2028), SLIGKV at 50 µM (PAR2-AP, Sigma, S9192), polygodial at 3 µM (Santacruz, sc-201489), and histamine at 1 mM (Sigma, H7125) to evaluate the functionality of the receptor and the capacity of differentiated cells to induced a Ca2+ signal following stimulation. For each agonist, a random field was recorded. Each activation by an agonist was recorded in a compartment that was distinct from other activations.
6
Endothelial VWF Secretion Assay
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Endothelial cells were grown in 6-well wells or on 24-mm polyester Transwell membranes with 0.4-µm pores (3450, Costar) and cultured at full confluence for 4 to 5 days. Unstimulated VWF release was determined in conditioned EGM-18 medium after 24-hour incubation. Stimulated VWF release was assayed after a 15-minute preincubation in serum-free medium M199 (ThermoFisher, 22340) supplemented with 0.2% (wt/vol) BSA (Merck, 112018). Cells were stimulated in serum-free medium supplemented with 0.1 to 100 µmol/L histamine (Sigma-Aldrich, H7125), 10 µmol/L forskolin (Sigma-Aldrich, F6886) with 100 µmol/L IBMX (Sigma-Aldrich, I7018), or vehicle (unstimulated) for 30 minutes, unless stated otherwise. Lysates were made in serum-free media supplemented with 1% Triton X-100 and protease inhibitors. Polarized VWF secretion was assayed essentially as described,25 (link) and conditioned media were collected separately from the top (apical) and bottom compartment (basolateral). VWF and VWFpp levels were determined by ELISA.
7
Ionomycin and Histamine Stimulation Protocol
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For ionomycin/histamine treatments, 5 × 105 cells were seeded in 35 mm dishes. The following day, regular growth medium was replaced by 1 ml of live-cell medium (DMEM with 25 mM d -glucose, 4 mM d -glutamine, and 25 mM HEPES, supplemented with 10% newborn calf serum). Another 1 ml of live-cell medium containing 8 µM ionomycin (I0634; Sigma; from a 1 mM stock in DMSO) was added swiftly in a circular motion onto the plate, making the final ionomycin concentration 4 µM. The same procedure was applied for histamine treatments, with a final concentration of 100 µM (H7125; Sigma; from a 100 mM stock in DMSO). Stimulations were carried out for the indicated time durations (Figure 1, B and E ), followed by instant medium removal and cell lysis for Western blotting.
8
Inducing Facial Itch Responses
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Pruritogens and algogen (histamine, H7125; chloroquine (CQ), C6628; capsaicin, M2028) were purchased from Sigma-Aldrich (St. Louis, MO, USA). To induce itch responses in the facial region, histamine (50 μg) and chloroquine (40 μg) were dissolved in 10 μL of PBS, and capsaicin (10 μg) dissolved in 10 μL of solution (7% Tween 80:20% ethanol:73% PBS) was intradermally injected into the cheek region as reported previously (26 (link)–28 (link)).
9
Intravital Imaging of Mouse Ear Vasculature
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Intravital imaging of the mouse ear with intradermal injection has been described previously (Honkura et al., 2018 (link)). Briefly, following systemic administration of 2000 kDa FITC (SigmaAldrich, FD2000S) or TRITC Dextran (ThermoFischer Scientific, D7139) by tail-vein injection, mice were sedated by intraperitoneal injection of Ketamine-Xylazine (120 mg/kg Ketamine, 10 mg/kg Xylazine) and the ear secured to a solid support. Mice were maintained at a body temperature of 37 °C for the entire experiment, maximum 90 min. Time-lapse imaging was performed using single-photon microscopy (Leica SP8). For intradermal EC stimulation, a volume of approximately 0.1 μl histamine (SigmaAldrich, H7125), concentration 10 ng/μl, was injected using a sub-micrometer capillary needle. 10 kDa TRITC Dextran (ThermoFischer Scientific, D1817) was used as a tracer. Leakage sites were identified in time-lapse imaging as defined sites of concentrated dextran in the extravascular space .
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