All reagents were purchased from Sigma-Aldrich unless otherwise specified. Bisindolylmaleimide I was purchased from Cayman Chemical. [3H]glucosamine was purchased from PerkinElmer. All fluorescent reagents, including WGA-Alexa 647, CFSE, and CellMask Deep Red, and secondary antibodies were from Thermo. Caspase 3, caspase 9, and PARP were from Cell Signaling. Enzyme-linked immunosorbent assay (ELISA) DuoSet kits for IL-1β, IL-1α, and TNF-α as well as the anti-Vamp8 antibody were from R&D. VAMP8-pHluorin was a kind gift from Rytis Prekeris (University of Colorado), and pmRuby2-MUC2CK was synthesized by Genscript as previously described (15 (link), 20 (link), 22 (link)). Mouse 41-plex cytokine arrays were constructed by Eve Technologies on the basis of tissue lysates normalized to 1 mg/ml.
3h glucosamine
Manufactured by PerkinElmer
3H-glucosamine is a radiolabeled compound used in various research applications. It contains the radioactive isotope tritium (3H) attached to the glucosamine molecule. This compound is commonly used as a tracer to study biological processes involving glucosamine, such as glycosylation and cartilage formation.
Automatically generated - may contain errors
Lab products found in correlation
Cellmask deep red, by Thermo Fisher Scientific (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Bisindolylmaleimide 1, by Cayman Chemical (1 mentions)
Wga alexa 647, by Thermo Fisher Scientific (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Kaiser test reagents, by Merck Group (1 mentions)
Cytochalasin b, by Merck Group (1 mentions)
Dmem f12, by Thermo Fisher Scientific (1 mentions)
Cytochalasin d, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
4 protocols using 3h glucosamine
1
Comprehensive Molecular Analysis of Cell Signaling
2
Glycosylation Dynamics in MDA-MB-231 Cells
3
Measuring Glycosaminoglycan Synthesis in MPS Fibroblasts
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MPS fibroblasts were grown in normal medium supplemented with 7 μCi/mL 3H-glucosamine (PerkinElmer) up to 80% confluence. The radioactive medium was removed, and cells were incubated for 24, 36, and 48 hr in normal medium containing 2% FBS and 0, 5 × 10−8, 10−7, 5 × 10−7, 10−6 M NK1. Cells were washed with PBS before harvesting, suspended in water, and lysed using freeze-thaw cycles. An aliquot of cell lysate was taken out for the determination of protein concentration using the Lowry method.69 Lipids were extracted by the addition of chloroform and methanol (chloroform-methanol-water 4:8:3, v/v/v). After 10-min of incubation at room temperature, extracts were recovered by centrifugation (10,000 × g for 10 min), washed with acetone, dried, and subjected to proteolysis overnight at 65°C with 1 mg/mL papain in 100 mM sodium acetate buffer containing 5 mM EDTA and 5 mM cysteine (pH 5.5). The content of 3H-glucosamine was measured by liquid scintillation counting and normalized against protein concentration.
4
Quantifying Mucin Secretion in Murine Colitis
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To quantify mucin secretion in vivo, mice were fasted overnight and injected intraperitoneally with 20 μCi of 3H-glucosamine (PerkinElmer, Waltham, MA) in PBS for 3h to metabolically label newly synthesized mucin into galactose, N-acetyl-glucosamine and N-acetyl-D-galactosamine in the mucin monomer as described previously [16 (link),50 (link)]. Colonic loops were used as a model for short-term infection studies (3h after infection), as described previously [51 (link)]. Briefly, Muc2+/+ and Muc2−/− mice were anesthetized with isoflurane inhalant anesthesia (Pharmaceutical Partners of Canada, Richmond Hill, ON). A laparotomy was performed, and the colon was exteriorized and ligated with 3–0 black silk sutures (Ethicon, Somerville, NJ; Peterborough, ON, Canada) at the proximal end and ~2 cm below. Care was taken to keep the mesenteries, blood vessels, and nerves intact. Virulent log-phase Eh trophozoites (1 × 106) in 100 μL PBS (pH 7.3) were inoculated into the loop. To quantify secretion of high molecular weight (Vo) mucin and non-mucin components, secreted 3H-labeled glycoproteins were fractionated using a Sepharose 4B columns as described previously [16 (link),50 (link)].
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