Principle
This kit can detect glucose found within cell culture medium or intracellular glucose by measuring the absorbance of the colored WST formazan dye. The degree of absorbance depends on the amount of glucose present within the sample.This kit contains a glucose standard solution that can be used to create a standard curve. This allows for the quantitation of glucose levels present in the sample.
Procedure
Procedures are so easy that you simply incubate the plate after the addition of culture supernatant or tissue/cell lysates prior to adding a reagent.
Preparation of Standard Curve
Glucose levels in a sample can be measured by a calibration curve established with Glucose Standards included in this kit. If the glucose levels are greater than or equal to 0.5 mmol/L, the sample must be diluted before measurement.
Measurement of Glucose level in combination with Lactate Assay Kit
By using Glucose Assay Kit-WST and Lactate Assay Kit-WST, we have successfully measured metabolic activity changes of phloretin, a protein transport inhibitor, when it is added to Jurkat cells.
Precautions when using this kit.
Cell counts may need to be normalized. When cells are analyzed in a microplate, the results obtained may sometimes differ depending on cell numbers per well.In such cases, normalization of the measured values obtained from cell counting and total protein will be necessary.In this kit, cell numbers can be easily measured by the fluorescence intensity induced by a reagent added to cell culture medium for staining nuclei.
Q. Precautions when measuring samples other than cell culture supernatants
A. Samples other than cell culture supernatants, such as cell lysates and plasma, contain proteins and can result in the increased background fluorescence. Therefore, we recommend to remove proteins in the sample. For details, please refer to the Q&A
"Can I measure the intracellular glucose?”.
Q. I do not have a 450 nm filter. What other filters can I use?
A. You can use filters with an absorbance between 450 nm and 490 nm. However, the absorbance value can become lower if the sample is not measured at 450 nm.
Q. Can I quantify L-Glucose levels?
A. No, the kit is used for the quantification of β-D-Glucose levels.
Q. How many samples can I measure?
A.
* The number of samples that can be recorded when the standard curve and sample is measured in triplicates.
*A plate layout for Glucose standard solution and sample
Q. Can I measure the intracellular glucose?
A. Yes. You can prepare your sample as below for example.
(1) Collect cells
*1 into 1.5 ml microtube.
*The number of cells required for measurement depends on the cell type.
The figures below show the results of the measurement in HepG2 cells and Jurkat cells.
(2) Centrifuge at 300 x g for 5 minutes and remove the supernatant.
(3) Add 300 µl of cold PBS, suspend by pipetting, centrifuge at 300 x g for 5 minutes,
and remove the supernatant.
(4) Add 250 µl of Cell Lysis Solution
*2 (0.1% Triton-X) and lyse the cells by pipetting.
Centrifuge the cells at 12,000 x g for 5 minutes.
(5) Transfer 200 µl of the supernatant from step (4) to a 10K ultra centrifugal filter and centrifuge at 12,000 x g for 10 minutes.
*If you measure with n=3, in total 150 µl or more sample is required (50 µl per well × 3 wells).
*If the filtrate is not left more than 150 µl after centrifugation, centrifuge it longer.
(6) Use the filtrate obtained in step (5) as a measurement sample.
Then, measure the glucose concentration according to the product manual supplied with the kit.
*If necessary, dilute the test sample with cell lysate to keep it within the calibration curve range (0-0.5 mmol/l).
*1) In order to detect glucose levels of more than 0.02 mmol/l, you need at least 1 x 10
5 cells HepG2 cells or 2 x 10
6 Jurket cells.
*2) You cannot use buffers containing SDS because SDS in the cell lysate inhibits coloring of the dye in the kit.
