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A complete reagent set to measure 2DG in cells after cell lysis by sonication

Glucose Cellular Uptake Measurement Kit (Broad Range, Fluorometric)

Background

Insulin is a hormone that lowers the blood glucose level and is known to help glucose enter cells through an effect that enhances the activity of glucose transport, primarily in insulin-sensitive organs cells, such as adipocytes and skeletal muscles.

The effect of insulin and other growth factors on cells is often assessed in the laboratory by measuring glucose uptake activity. Typically, glucose uptake is measured using 3-O-Methyl-D-Glucose or 2-deoxyglucose (2DG) labeled with radioactive 3H or other isotope. However, the use of radioactive isotopes is not available to all labs and is subject to many restrictions. This assay kit was developed to provide a simple, rapid and convenient means to measure cellular glucose uptake without the use of radioisotope.

This assay utilizes the glucose analog 2-deoxyglucose in place of glucose. Like glucose, 2DG taken up by cells is rapidly phosphorylated by hexokinase to 2-deoxyglucose-6-phosphate (2DG6P). However, unlike glucose, 2DG6P is not further metabolized and accumulates in cells. Using the provided reagents, cell lysates are then assayed for 2DG6P levels in a coupled enzymatic re-dox reaction that produces a fluorescent signal whose intensity is proportional to the amount of accumulated 2DG6P. 2DG levels in cell lysate samples are thus calculated by comparing their fluorescence intensity to a standard curve produced with known amounts of 2DG6P.

Feature and Advantages

•Assay measures amount of 2-deoxyglucose (2DG) (Glucose analog) uptake. 
•Like glucose, 2DG taken up by cells is rapidly phosphorylated by hexokinase to 2-deoxyglucose-6-phosphate (2DG6P). However, 2DG6P is not further metabolized and accumulates in cells. 
•Cell lysates are assayed for 2DG6P levels in a coupled enzymatic re-dox reaction that produces a fluorescent signal whose intensity is proportional to the amount of accumulated 2DG6P. 
•2DG levels in cell lysate samples are calculated by comparing their fluorescence intensity to a standard curve produced with known amounts of 2DG6P. 

  Glucose Cellular Uptake Measurement Kit, Broad Range, Fluorometric 
(Cat. No. MBR-PMG-K01E)
【Related Product】
2-Deoxyglucose (2DG) Uptake Measurement Kit
Cat. No.: CSR-OKP-PMG-K01E
Assay Format Non-radioactive Non-radioactive
Assay Time 3 hours 5 ~ 7 hours (2 Days)
Detection Method Fluorometric (Ex 540nm/Em 590nm) Chromogenic  (420nm)
Measurement Range Broad  ( 0-50μM 2DG6P) High sensitivity (0 to 5μM 2DG6)
Features Fast, convenient, single step suitable for high sample through put.。 Sensitivity comparable with radioactive assays. High accuracy. High precision.

Kit component

Reagent Size Quantity Storage conditions for opened vials
2DG6P Solution (1 mM) 500 μL 3  x 3 ml

-20°C

(protect from light)

Sample Diluent Buffer Concentrate (100x) 5 ml 1 vial
Substrate Buffer 9 mL 1 vial
Fluorescent Substrate 120 μL 1 vial
Enzyme Solution 270 μL 1 vial

Reagents for 100 reactions are supplied. 
Store kit at -20°C until expiration date printed on the label, protected from light.

Materials required but not supplied.

● Ultrapure water (distilled water) 
● 2-deoxyglucose suggested product 
● Bovine Serum Albumin suggested product 
● 96-well black wall microplate 
● 96-well fluorescent plate reader 
● Suitable sonication device for cell lysis.

Application Data

2DG uptake by insulin-stimulated adipocytes following the differentiation of 3T3-L1 cells in culture

   

Example of insulin stimulation and sample preparation using adipocytes differentiated from 3T3-L1 cells in culture.

 

For your own experiments with your own cells, it will be necessary to adjust and optimize all cell culture media and conditions, reagent concentrations, incubation times, and other parameters according to your experimental needs. 

The following procedure is for 3T3-L1 cells grown in one well of a standard 12-well culture plate.

Reagents and Media

・Serum-free culture medium
・Krebs Ringer Phosphate HEPES (KRPH) buffer at 37°C
(1.2mM KH2PO4, 1.2mM MgSO4, 1.3mM CaCl2, 118mM NaCl, 5mM KCl, 30mM Hepes, pH7.5)
・BSA (essentially fatty acid free and globulin free grade, use Sigma Ca. No. A0281 or equivalent)
・2-Deoxy-D-glucose (2DG) solution
・ Insulin solution
・ PBS(-)
・Phloretin or Cytochalasin B (or other glucose uptake inhibitor)

Cell Preparation

1) Differentiate 3T3-L1 cells to adipocytes in a 12-well culture plate.
2) Replace culture media with serum-free medium. Return to incubator for 6 hours.
3) Gently wash cells 3 times with 1.5mL of warm KRPH buffer without BSA?.
4) Gently add 3mL of warm KRPH buffer containing 2% BSA.

Add insulin.

5) For this example, insulin is added to a final concentration of 1μM .
6) Incubate cells for 18 min at 37°C.

Add 2DG.

7) For this example, 2DG is added to a final concentration of 1mM.
8) Incubate cells at 37 C for 20 minutes.
9) Wash cells gently 3x with cooled PBS containing 200μM Phloretin to inhibit further 2DG uptake.

Cell Lysis and Sample Preparation

10) Add 1.5mL of 1x Sample Diluent Buffer.and lyse cells by sonication with a microtip sonicator.
11) Collect cell lysate to tube, and apply heat treatment at 80°C for 15 minutes.

Note: Do not add protease inhibitors to cell lysates.
Note: If an alternate method for cell lysis will be used, do not use NaOH as NaOH degrades 2DG6P.

12) Centrifuge lysates at 15000 x g for 20minutes at 4°C
13) Transfer cell lysate supernatants to a fresh tube. These cell lysate supernatants are your experimental samples. 

Note: Supernatants can be stored at -20°C for later analysis.

Measure 2DG in Cell Lysis Supernates

 14) Follow the procedure of [Section III-1] “2DG Measurement Protocol” above.

 

Product List

Product Name Cat# Quantity Price

Glucose Cellular Uptake Measurement Kit (Broad Range, Fluorometric)

CSR-MBR-PMG-K01E 100TEST

¥ 105,600
$ 1408
€ 1056

References

> Yamamoto N, et al ,(2006). A nonradioisotope, enzymatic assay for 2-deoxyglucose uptake in L6 skeletal muscle cells cultured in a 96-well microplate. Anal. Biochem. 351: 139-145

To be used for research only. DO NOT use for human gene therapy or clinical diagnosis.