葡萄糖实验方案 | 糖类 | 生物化学

Quantifying Intestinal Glucose Absorption Using Isolated Vascularly Perfused Rat Small Intestine

利用离体灌流大鼠小肠模型定量分析肠道葡萄糖吸收

CB Cecilie Bæch-Laursen

SB Sabine Bæch-Laursen

JH Jens Juul Holst

0 Q&A

1757 Views

Nov 20, 2025

Intestinal glucose absorption has been studied for several decades. However, the different methods available for investigating absorption are often the reason for variability in the results, and it is difficult to measure the relative contribution of paracellular absorption using existing methods. Thus, we have established a new model for measuring glucose absorption. In the isolated in situ vascularly perfused small intestine, the intestinal epithelium is completely preserved, and the entire transport pathway is intact. In the present model, we use radioactive labeled ¹⁴C-d-glucose, which allows for sensitive quantification of glucose absorption even with low luminal concentrations. The described method is optimized for intestinal glucose absorption but can be applied to other macro/micronutrients that can be radioactively labeled. The described procedure is a novel approach for measurements of intestinal nutrient absorption and gut permeability in which luminal nutrient concentrations resemble physiological concentrations.

Slot Blot Analysis of Intracellular Glyceraldehyde-Derived Advanced Glycation End Products Using a Novel Lysis Buffer and Polyvinylidene Difluoride Membrane

新型裂解缓冲液和聚偏二氟乙烯膜在细胞内甘油醛衍生晚期糖基化终产物的点杂交分析中的应用

TT Takanobu Takata

HM Hiroki Murayama

TM Togen Masauji

0 Q&A

1977 Views

Jul 20, 2024

Advanced glycation end products (AGEs) are formed through the reaction/modification of proteins by saccharides (e.g., glucose and fructose) and their intermediate/non-enzymatic products [e.g., methylglyoxal and glyceraldehyde (GA)]. In 2017, Dr. Takanobu Takata et al. developed the novel slot blot method to quantify intracellular GA-derived AGEs (GA-AGEs). Although the original method required nitrocellulose membranes, we hypothesized that the modified proteins contained in the AGEs may be effectively probed on polyvinylidene difluoride (PVDF) membranes. Because commercial lysis buffers are unsuitable for this purpose, Dr. Takata developed the slot blot method using an in-house-prepared lysis buffer containing 2-amino-2-hydromethyl-1,3-propanediol (Tris), urea, thiourea, and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) that effectively probes AGEs onto PVDF membranes. The slot blot method also entails the calculation of Tris, urea, thiourea, and CHAPS concentrations, as well as protein and mass to be probed onto the PVDF membranes. GA-AGE-modified bovine serum albumin (BSA, GA-AGEs-BSA) is used to draw a standard curve and perform neutralization against a non-specific combination of anti-GA-AGEs antibodies, thereby enabling the quantification of GA-AGEs in cell lysates. This paper presents the detailed protocol for slot blot analysis of intracellular GA-AGE levels in C2C12 cells.

Isolation of Tumor Cells Based on Their Distance from Blood Vessels

基于与血管距离的肿瘤细胞分离

0 Q&A

6172 Views

May 20, 2020

Differential exposure of tumor cells to microenvironmental cues greatly impacts cell phenotypes, raising a need for position based sorting of tumor cells amenable to multiple OMICs and functional analyses. One such key determinant of tumor heterogeneity in solid tumors is its vasculature. Proximity to blood vessels (BVs) profoundly affects tumor cell phenotypes due to differential availability of oxygen, gradient exposure to blood-borne substances and inputs by angiocrine factors. To unravel the whole spectrum of genes, pathways and phenotypes impacted by BVs and to determine spatial domains of vascular influences, we developed a methodology for sorting tumor cells according to their relative distance from BVs. The procedure exemplified here using glioblastoma (GBM) model is based on differential uptake of intra-venously injected, freely-diffusing fluorescent dye that allows separation of stroma-free tumor cells residing in different, successive microenvironments amenable for subsequent OMICs and functional analyses. This reliable, easy to use, cost effective strategy can be extended to all solid tumors to study the impact of vasculature or the lack of it.

Single-run HPLC Quantification of Plant Cell Wall Monosaccharides

Single-run HPLC定量分析植物细胞壁单糖

AM Alexandra Menna

MF Michaela Fischer-Stettler

BP Barbara Pfister

GS Gloria Sancho Andrés

DH Duncan Holbrook-Smith

CS Clara Sánchez-Rodríguez

0 Q&A

5308 Views

Mar 5, 2020

The plant cell wall is a complex network of polysaccharides and proteins that provides strength and structural integrity to plant cells, as well as playing a vital role in growth, development, and defense response. Cell wall polysaccharides can be broadly grouped into three categories: cellulose, pectins, and hemicelluloses. Dynamic interactions between polysaccharides and cell wall-associated proteins contribute to regions of flexibility and rigidity within the cell wall, allowing for remodeling when necessary during growth, environmental adaptation, or stress response activation. These polysaccharide interactions are vital to plant growth, however they also contribute to the level of difficulty encountered when attempting to analyze cell wall structure and composition. In the past, lengthy protocols to quantify cell wall monosaccharides contributing to cellulose as well as neutral and acidic cell wall polysaccharides have been used. Recently, a streamlined approach for monosaccharide quantification was described. This protocol combines a simplified hydrolysis method followed by several runs of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Here, we present an updated version of this protocol in which we can analyze all nine cell wall monosaccharides in a single high-performance liquid chromatography HPAEC-PAD gradient profile. The inclusion of an enzymatic starch degradation, as well as alternate internal standards for added quantification accuracy, and a ready-to-use Python script facilitating data analysis adds a broadened scope of utility to this protocol. This protocol was used to analyze Arabidopsis light-grown seedlings and dark-grown hypocotyls, but is suitable for any plant tissues.

Steady-state and Flux-based Trehalose Estimation as an Indicator of Carbon Flow from Gluconeogenesis or Glycolysis

以基于稳态和通量的海藻糖测定作为糖异生或糖酵解碳流指标

Ritu Gupta

SL Sunil Laxman

0 Q&A

5660 Views

Jan 5, 2020

Trehalose (and glycogen) is a major storage carbohydrate in many cells, including S. cerevisiae. Typically, trehalose (a disaccharide of glucose) is synthesized and stored through gluconeogenesis. However, trehalose can also be made directly from glucose, if glucose-6-phosphate is channeled away from glycolysis or pentose phosphate pathway. Therefore, analyzing trehalose synthesis, utilization or its accumulation, can be used as a sentinel read-out for either gluconeogenesis or rewired glucose utilization. However, the steady-state measurements alone of trehalose cannot unambiguously distinguish the nature of carbon flux in a system. Here, we first summarize simple steady-state enzymatic assays to measure trehalose (and glycogen), that will have very wide uses. Subsequently, we describe methods of highly sensitive, quantitative LC-MS/MS based to measure trehalose. We include methods of ¹³C stable-isotope based pulse-labeling experiments (using different carbon sources) with which to measure rates of trehalose synthesis, from different carbon metabolism pathways. This approach can be used to unambiguously determine the extent of carbon flux into trehalose coming from gluconeogenesis, or directly from glucose/glycolysis. These protocols collectively enable comprehensive steady-state as well as carbon flux based measurements of trehalose. This permits a dissection of carbon flux to distinguish between cells in a gluconeogenic state (conventionally leading to trehalose synthesis), or cells with rewired glucose metabolism (also leading to trehalose synthesis). While the methods presented are optimized for yeast, these methods can be easily adapted to several types of cells, including many microbes.

FACS-based Glucose Uptake Assay of Mouse Embryonic Fibroblasts and Breast Cancer Cells Using 2-NBDG Probe

使用2-NBDG探针并基于FACS的小鼠胚胎成纤维细胞和乳腺癌细胞的葡萄糖摄取测定

SD Shengli Dong

Suresh K Alahari

0 Q&A

11757 Views

Apr 20, 2018

This is a flow cytometry-based protocol to measure glucose uptake of mouse embryonic fibroblasts (MEFs) and breast cancer cells in vitro. The method is a slightly modified and updated version as previously described (Dong et al., 2017). Briefly, the target cells are incubated with the fluorescently tagged 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG) for 2 h or 30 min, and the efficiency of glucose uptake is examined using a flow cytometer. This method can be adapted to measure a variety of adipocytes, immune cells, MEFs and cancer cells.

Determination of Intra- and Extracellular Glucose in Mycelium of Fusarium oxysporum

尖孢镰刀菌菌丝体的体内和体外葡萄糖的测定

Carmen Ruiz-Roldan

M. Isabel G. Roncero

0 Q&A

11066 Views

Jul 20, 2016

To study alterations in the metabolism and/or in the transport of glucose during Fusarium oxysporum vegetative growth, we determined intracellular glucose levels in different fungal strains, as well as the amount of glucose remaining in the supernatants after growth in synthetic medium (SM) supplemented with either 0.05 or 2.5% glucose. We used the Glucose (GO) Assay Kit (Sigma-Aldrich) following the instructions of the manufacturer with some modifications. The protocol described here can be applied to other filamentous fungi.

¹³C Tracer Studies of Metabolism in Mouse Tumor Xenografts

13C 示踪研究老鼠肿瘤异种移植中的新陈代谢

AL Andrew N. Lane

JY Jun Yan

TF Teresa W-M. Fan

0 Q&A

13029 Views

Nov 20, 2015

Mice are widely used for human tumor xenograft studies of cancer development and drug efficacy and toxicity. Stable isotope tracing coupled with metabolomic analysis is an emerging approach for assaying metabolic network activity. In mouse models there are several routes of tracer introduction, which have particular advantages and disadvantages that depend on the model and the questions addressed. This protocol describes the bolus i.v. route via repeated tail vein injections of solutions of stable isotope enriched tracers including ¹³C₆-glucose and ¹³C₅,¹⁵N₂-glutamine. Repeated injections give higher enrichments and over longer labeling periods than a single bolus. Multiple injections of glutamine are necessary to achieve adequate enrichment in engrafted tumors.

Analysis of Malondialdehyde, Chlorophyll Proline, Soluble Sugar, and Glutathione Content in Arabidopsis seedling

拟南芥中丙二醛、叶绿素脯氨酸、可溶性糖和谷胱甘肽含量的分析

Zhijin Zhang

Rongfeng Huang

2 Q&A

35384 Views

Jul 20, 2013

The protocol has four sub-protocols, which are about the measurement of malondialdehyde, chlorophyll proline, soluble sugar, and glutathione content, respectively, in Arabidopsis seedling by using spectrophotometer. These methods are simple, effective and reproducible, which will help the researchers who are not familiar with these approaches, quickly get reliable results.

Glucose Production Assay in Primary Mouse Hepatocytes

原代小鼠肝细胞中的葡萄糖生成分析

Michihiro Matsumoto

MS Mashito Sakai

4 Q&A

27252 Views

Nov 5, 2012

Hepatic glucose production is a primary determinant of fasting hyperglycemia in type 2 diabetic patients. Glucagon-cAMP-PKA pathway increases, but insulin-PI3 kinase-Akt pathway suppresses glucose production. This assay aims to evaluate the ability of isolated mouse hepatocytes to release newly synthesized glucose mainly from lactate and pyruvate as the substrates (i.e. gluconeogenesis) under basal, cAMP-, or cAMP plus insulin-treated condition.