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2-Deoxyglucose (2DG) Uptake Measurement Kit Frequently Asked Questions


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Question Can the kit be used to measure 2-DG uptake in micro-organisms? Are there any experimental examples with microorganisms?
Answer Unfortunately, we don't have any experience measuring 2-Deoxyglucose uptake with microorganisms so we do not know whether it is possible to do the measurement in micro-organisms.
Question

Is there any experimental data for glucose uptake in E.coli?

Answer
Unfortunately, we don't have any experience measuring 2-Deoxyglucose uptake with E. coli.
Please confirm 2-deoxyglucose is taken into bacteria and the -deoxyglucose-6-phosphate remains in vivo in advance.
If you have information or experimental experience, this kit would work for the measurement.
Unfortunately, we don't have any experience measuring 2-Deoxyglucose uptake with E. coli. Please note that 2-deoxyglucose is taken into bacteria and the -deoxyglucose-6-phosphate remains in vivo in advance. If you have information or experimental experience, this kit would work for the measurement.
Question What type of 96-well plate should be used with the kit? A white plate or a Clear plate?
Answer Please use transparent plates. There isn't a particular plate type we strongly recommend.
Question How do I analyze the data for the 2DG assay? I don't understand the unit for the standard curve is microM, but how do I get pmole/well as shown in the example graph in the the protocol?
Answer The amount of 2DG6P in this kit is shown by molar concentration [Molarity]. Please contact us for details of a measurement example and the calculation example in our laboratory. 
Question Regarding the process of sonication. Please tell me the parameter setup of microtip and sonication.                   
Answer This kit is a kit that examines uptake of glucose into the cell by measuring 2DG6P uptaken in the cell. The procedure (page-6 (9)) is a procedure for obtaining cell lysate by disrupting the cell. The method (parameter setup) of obtaining cell lysate in sonication device might be different depending on the model. Please follow the recommendation of the model about the setting and the procedure. When they are uncertain, please confirm the cell disrupting with the microscope.
Question Have there been any tests carried out cancer cells and tumours?
Answer We do not have experimental examples for Cancer cell lines and Carcinoma. 
Question Have you used the 2DG uptake assay in animals to look at uptake in tumors?  I want to remove the tumors, section them, and see where glucose is taken up.
Answer Unfortunately we do not currently have any information about uptake in tumors for in vivo use with our current kit. However we are concentrating on developing the in vivo kit now. And we have had reports that some customers made their own modifications to the in vitro kit including adding hexokinase to use for their in vivo experiments, but we do not have established protocol as well.
Question Regarding the 2DG uptake measurement kit, is it possible to use half the volumes of the reagents instead (e.g. a total volume of 100ul  instead of 200 ul per reaction)?How will this influence the performance of the kit? 
Answer We are sorry, but we can not guarantee the experiment result which is saved or used half volumes. It may change the reactivity by dimension or instrument. And it also will stable the measurement, thus we strongly recommend you test according to the protocol.
Question Please provide the solvent of the 1mM 2DG6P solution of your 2DG-Uptake kit.
Answer We cannot provide you with a specific components, but our 1mM 2DG is dissolved in distilled water.
Question What is the solution A component?
Answer

Unfortunately, we cannot provide you information of the components.

Question The protocol calls for the use of a microtip sonicator for cell lysis, which is not available to our lab. Is the sonication step (step 9) required or is there another way to lyse the cells that would be acceptable for your assay?
Answer

If your lab does not have a microtip sonicator, there is an alternative way to make samples solubilize. When you lyse cells, please use TritonX-100 i1“j with less volume. Please dilute the extract solution to 10-20 times. 

This diluted solution will be used as a sample. You may feel this dilution ratio is high, but this solubilization needs less surfactant i1~2mljcompared with the procedure by using microtip sonicato (3mL as described in the protocol). Therefore, final sample concentration will be the same as when you use microtip sonicator. Please don't use SDS as a surfactant because it will inhibit the reaction system of this kit.

Question I have another question regarding the buffer used for cell lysis: should it contain sodium chloride and  chelators in addition to the Tris-HCL and Triton 100-x, or is it better to leave these out?
Answer

We recommend you don't add sodium chloride and chelators, as the reaction system will be affected negatively when it increases concentration

Question Can I use the 2-DG kit for mature adipocytes? Is there any effect on suspension cells?
Answer

Mature adipocytes can be used for this kit. Mature adipocytes are easy to peel from surface of the plate. So your customer’s cell condition is natural. For use of this kit by using mature adipocyte, cell floating gives no effect. However, mature adipocyte is difficult to submerge with centrifugation, and strong centrifugation makes damage to the cells. So, please be careful when the assay will be performed. If the customer worries about the damage to the cells by centrifugation, there is another choice. By using the culture plate coating with type-I collagen or fibronectin, the adhesiveness of the mature adipocyte becomes strong.

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