The following primary antibodies were used for immunofluorescence staining: anti-Slo1 (UC Davis/NIH NeuroMab Facility, Davis, CA or Abcam, Cambridge, UK), anti-Lamp1 (H4A3, 1D4B, Developmental Studies Hybridoma Bank, USA), Alexa 488 goat anti-rat antibody, Alexa 488 goat anti-rabbit antibody, Alexa 546 goat anti-mouse antibody and Texas Red goat anti-mouse antibodies were purchased from Invitrogen (Invitrogen, USA). The following chemicals were also used: Paxilline (Cayman Chemical Company, USA); TEA (Sigma, USA); ML-SA1 (Tocris Bioscience, UK); ML-SI1 (Enzo Life Sciences Inc, USA); NS1619 (Sigma, USA); BAPTA-AM (Tocris Bioscience, UK); EGTA-AM (Invitrogen, USA); Recombinant Mouse M-CSF (carrier-free) (BioLegend, USA); 4-Methylumbelliferyl N-acetyl-β-d-glucosaminide (Sigma, USA).
4 methylumbelliferyl n acetyl β d glucosaminide
Manufactured by Merck Group
Sourced in United States, Germany
4-methylumbelliferyl N-acetyl-β-D-glucosaminide is a fluorogenic substrate used for the detection and quantification of N-acetyl-β-D-glucosaminidase (NAG) activity. NAG is an enzyme involved in the degradation of glycosaminoglycans and is commonly used as a biomarker for various conditions.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (4 mentions)
C48 80, by Merck Group (3 mentions)
4 methylumbelliferyl β d glucopyranoside, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Sparkcontrol magellan plate reader, by Merck Group (2 mentions)
Fluo 3 am ester, by Biotium (2 mentions)
Tyrode buffer, by Merck Group (2 mentions)
96 well cell culture plate, by Corning (2 mentions)
Victor plate reader, by PerkinElmer (2 mentions)
Synergy htx fluorimeter, by Agilent Technologies (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
4 methylumbelliferyl β d glucuronide, by Merck Group (2 mentions)
Paxilline, by Cayman Chemical (1 mentions)
Alexa 488 goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Ml sa1, by Bio-Techne (1 mentions)
Ml si1, by Enzo Life Sciences (1 mentions)
Ns1619, by Merck Group (1 mentions)
Bapta am, by Bio-Techne (1 mentions)
Egta am, by Thermo Fisher Scientific (1 mentions)
Recombinant mouse m csf carrier free, by BioLegend (1 mentions)
33 protocols using 4 methylumbelliferyl n acetyl β d glucosaminide
1
Immunofluorescence Staining of Slo1 and Lamp1
2
RBL Cell Mediator Release Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RBL cell mediator release assay was performed as described elsewhere [19 (link)]. Briefly, RBL 2H-3 (ATCC, Manassas, VA, USA) cells were plated into 96-well plates (4 × 104 cells/well) and incubated with serum samples (1:300) from days −11 and 25 for 2 h at 37 °C in a 5% CO2 atmosphere. Cells were washed with Tyrode’s buffer (137 mM NaCl, 5.6 mM d -glucose, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM MgCl2, 0.4 mM NaH2PO4, 12 mM NaHCO3, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and 0.1% (w/v) BSA; pH = 7.4; Sigma-Aldrich) and degranulation of cells was induced by incubation with 0.3 µg/mL OVA in Tyrode’s buffer. Supernatants were analyzed for β-hexosaminidase content by incubation with 80 μM 4-methylumbelliferyl-N-acetyl-β-d-glucosaminide (Sigma-Aldrich) and measuring fluorescence at λex: 360 nm/λem: 465 nm with a SparkControl Magellan plate reader. Results show the percentage of total β-hexosaminidase release in serum samples compared to positive controls after adding 1% (v/v) Triton X-100 (Sigma-Aldrich) in double-distilled water.
3
Mast Cell Degranulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The rat mast cell line RBL‐2H3 was maintained in DMEM containing 10% FBS. For antigen‐triggered secretion, cells were sensitized by overnight incubation with 0.5 μg/ml monoclonal mouse anti‐DNP IgE (Sigma‐Aldrich #D8406) and secretion was stimulated with antigen (1 μg/ml DNP). Antigen was used to stimulate secretion, except in Fig EV1 A where 100 nM ionomycin (EMD Biosciences #407952) together with 100 nM PDBu (Sigma‐Aldrich #P1269) was used to stimulate secretion. Cells were stimulated in extracellular buffer (125 mM NaCl, 5 mM KCl, 20 mM HEPES, 1.5 mM MgCl2, 1.5 mM CaCl2, 10 mM d ‐glucose, pH 7.4). Secretion was assessed by measuring release of β‐hexosaminidase with 1.5 mM 4‐methylumbelliferyl N‐acetyl‐β‐d ‐glucosaminide (Sigma‐Aldrich #69585) in a fluorescent plate reader, Victor3 (PerkinElmer), at 37°C with RBL cells cultured in plastic 96‐well plates. β‐hexosaminidase levels were calculated by fitting a straight line to 11 measurement points acquired during 15 min. Cells were permeabilized with 0.5% Triton X‐100 to quantify β‐hexosaminidase retained in cells. Mean and standard error of the mean were calculated from multiple wells. In VAMP8 siRNA knockdown experiments, when comparing levels of secretion between stable cell lines expressing different VAMP8 mutants, secretion was normalized to siCtrl in the same stable cell line.
4
Mast Cell Degranulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SHL injection and its 2 intermediate fractions (ES and ELF), which were prepared according to the Chinese Pharmacopoeia23 were from Duoduo Pharmaceutical Co., Ltd. (Jiamusi, Heilongjiang, China). C48/80, 4-Methylumbelliferyl N-acetyl-β-D-glucosaminide, and Pluonic F-127 were purchased from Sigma-Aldrich (St Louis, MO, USA). Fluo-3 AM Ester and rhod-2/AM were from Biotium (San Francisco, CA, USA). Ketotifen and cromolyn sodium were from TCI (Tokyo, Japan) and National Institutes for Food and Drug Control (Beijing, China), respectively. Calcium Green-5N and PerCP-eFluor 710 labeled anti-mouse FcεR1 antibody were obtained from Invitrogen (Carlsbad, CA, USA) and eBioscience (San Diego, CA, USA), respectively. The transfection reagents Entranster TM-in vivo and -H4000 were from Engreen Biosystem (Beijing, China). Balb/c mice (male, 18–20 g) and SD rats (male, 160–180 g) were from Vital River Experimental Animal Services (Beijing, China).
5
β-Hexosaminidase Activity Assay in LG-EpCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
LG-EpCs (3 × 105/well) were cultured on pure dLG-HG, as well as on genipin/dLG-HG and DMSO/dLG-HG dilutions (200 mL/well), in 48-well cell culture plates for 7 days in duplicates of six biological replicates. β‐Hexosaminidase activity was measured as described previously.26 (link) In brief, cells were washed with serum-free DMEM and incubated with 300 µL serum-free DMEM for 2 hours (baseline value). Carbachol (Sigma-Aldrich) was added at a final concentration of 100 mM, and a stimulated sample was obtained after 30 minutes. For the measurement of β-hexosaminidase activity, 4-methylumbelliferyl N-acetyl-β-d -glucosaminide (Sigma-Aldrich) was used as a substrate. The fluorescence intensity was determined at 360-nm excitation and 450-nm emission (Victor X4 Multilabel Reader, PerkinElmer, Waltham, MA, USA).
6
Rat Basophil Leukemia Cell Degranulation
7
Measurement of OVA-Specific Antibody Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
OVA‐specific serum IgG1, IgG2a, IgE and IgA levels were determined by ELISA. The 96‐well microtitre plates were coated with OVA (5 μg ml−1). Serum samples were diluted 1:10,000 for IgG1, 1:100 for IgG2a, 1:10 for IgE and 1:10 for IgA. Rat anti‐mouse IgG1, IgG2a, IgE and IgA antibodies (2 μl ml−1; Pharmingen, San Diego, CA USA) were applied, followed by peroxidase‐conjugated mouse anti‐rat IgG antibodies (1:2000; Jackson Immuno Labs, West Grove, PA, USA). Antibody levels were quantified based on optical densities.
The allergen‐specific IgE levels in sera were quantified by analysing the degranulation of rat basophil leukaemia (RBL‐2H3) cells (Wiedermann et al.,2001 ). RBL‐2H3 cells were plated in 96‐well tissue culture plates (4 × 104 cells per well) and sensitized by incubation with mouse serum (diluted 1:270 or 1:810) for 2 h. The cells were washed, OVA (0.6 mg ml−1) was added, and the cells were incubated for 30 min at 37°C for degranulation. Supernatants were recovered and incubated with 4‐methylumbelliferyl‐N‐acetyl‐β‐D‐glucosaminide (Sigma‐Aldrich, St Louis, MO, USA), and β‐hexosaminidase was analysed using an Infinite M200 fluorescence microplate reader (λex:360 nm/λem:465 nm; Tecan Group, Grodig, Austria). The results are reported as the percentage of total β‐hexosaminidase released from cells after disruption with 1% Triton X‐100.
The allergen‐specific IgE levels in sera were quantified by analysing the degranulation of rat basophil leukaemia (RBL‐2H3) cells (Wiedermann et al.,
8
Quantification of Lysosomal Enzyme Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
iPS cell lysates or human fibroblasts prepared in 0.1 M citric buffer (pH 4.2) containing 0.1% Triton X-100 were assayed for β-hexosaminidase activity with 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma-Aldrich, St. Louis, MO) (16 (link)). Values were normalized using β-galactosidase activity and measured using 4-methylumbelliferyl β-D-galactopyranose (Sigma-Aldrich) (17 (link)). Activity was calculated as β-hexosaminidase activity normalized by β-galactosidase activity per minute and expressed as percentage of control cells.
9
Artemisia DPLP Basophil Degranulation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mediator release of purified DPLPs from Artemisia spp. was evaluated using a rat basophil degranulation leukemia (RBL) assay, as described in Reference [21 (link)]. RBL-2H3 cells, which were transfected with an antihuman IgE high-affinity receptor, were sensitized with patients’ sera overnight (n = 4). Cells were stimulated with increasing concentrations of the purified proteins (0.001–100 µg/mL), and the release of β-hexosaminidase was measured by the enzymatic cleavage of the fluorogenic substrate 4-methylumbelliferyl N-acetyl-β-D-glucosaminide (Sigma, Germany). Total release (100%) was obtained when cells were treated with 10% of Triton X-100, and mediator release of DPLPs is given as a percentage.
10
Quantifying Beta-Hexosaminidase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HFs were lysed in buffer containing 0.1% Triton X-100 and protease inhibitors. Five microliters (1–2 μg/µL protein) of sample was then incubated at 37 °C for 30 min in 100 µL of a solution containing 150 mM citrate, 0.2 M Na2HPO4 (pH 4.0) and 1.97 mM 4-methylumbelliferyl-N-acetyl-β-D-glucosaminide (Sigma-Aldrich, M2133), the substrate of β-hexosaminidase [26 (link)]. The reaction was immediately stopped by adding 190 µL of 0.3 M glycine-NaOH (pH 10.3). The β-hexosaminidase activity was measured with a TECAN infinite 200 PRO microplate reader (excitation: 360 nm; emission: 465 nm) and is represented as RFU/μg protein.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!