Hi all,
I have to do Glucose uptake assay using the MCF10A cell line using a fluorescence-based Glucose uptake assay kit (Cayman). Can anyone suggest what type of 96 well plates should be used? Do I need a black bottom plate or a black clear bottom plate?
I will be using a plate reader where light is coming from the top.
Can I see the cells seeded in a black bottom plate? As I need to check whether the cells have adhered or not and whether they have reached optimum density or not.
And finally, how long should I incubate the cells with 2-NBDG?
If anyone can share a detailed protocol of this experiment, it'll be really helpful.
Thanks in advance.
Swarnali
Hello Swarnali Kar ,
For this experiment I would recommend a black-walled, clear-bottom 96-well microplate. This setup minimizes well-to-well fluorescent contamination and autofluorescence and are ideal for fluorescence-based applications.
Regarding the time of incubation with 2-NBDG, unfortunately I do not have personal experience with MCF10A cells, but I can suggest you these manuscripts:
Article Development of High-Throughput Quantitative Assays for Gluco...
Article The cytotoxic effect and glucose uptake modulation of Baecke...
Good luck with your experiment!
Mario
Hi Swarnali, for fluorescence reading, I would suggest a black-walled clear bottom plate, as it reduces cross well fluorescent bleach and your light in reader is coming from upside down. I have incubated my primary cells with 2-NBDG for 30 min. After that, I have observed the cells for glucose uptake through both flow cytometry and CLSM. I have observed a very clear results. One thing I can suggest you is the optimization of concentration of 2-NBDG. Good luck.
Thank you very much, Mario Romani and Sanpreet Singh for your answers.
I definitely will standardize according to my cell line.
Can you please advise me about the highest and lowest concentration of 2-NBDG to treat the cells with? (As it is costly!!)
And can you suggest what steps to take after incubation with 2-NBDG is over? (Washing?)
And should I record data immediately after incubation is over or there is a time frame? (Actually, I have to go to another campus to use the plate reader and it takes around 30 mins).
Thanks again,
Swarnali
MCF10A cell lines are typically treated with 2-NBDG at a concentration of 100 uM.
For details, please refer to the following documents:
Population-Based Fluorometric Microplate Assays
To evaluate the kinetics of 2-NBDG uptake with increasing cell density, MCF10A and CA1d cell lines were seeded (0–30,000 cells/well) in clear-bottomed 96-well microplates in triplicate. Cells were allowed to adhere overnight at 37°C (for ~12 h) before performing uptake assays. After overnight incubation, all wells were washed twice with phosphate-buffered saline (PBS) and incubated with 2-NBDG (100 µM) for 10 min at 37°C in a humidified atmosphere of 5% CO2. We stopped the reaction by adding a twofold volume of ice-cold PBS and the wells were washed again with ice-cold PBS three times. The fluorescent signal before (autofluorescence) and after adding 100 µM 2-NBDG was measured using the fluorometric mode of a Victor-3 multi-well plate reader (using the 485 nmex and 520 nmemiss filter set). The net increase in fluorescence was normalized to the lowest signal (0 cells/well), which was taken as the ratiometric quantitation of 2-NBDG uptake in cells.
Reference:
Hassanein M, Weidow B, Koehler E, Bakane N, Garbett S, Shyr Y, Quaranta V. Development of high-throughput quantitative assays for glucose uptake in cancer cell lines. Mol Imaging Biol. 2011 Oct;13(5):840-52. doi: 10.1007/s11307-010-0399-5. PMID: 20809209; PMCID: PMC3627351.
If you think 2-NBDG is expensive, you can try following 2-NBDG.
https://www.glpbio.com/2-nbdg.html
Thank you Niki Yang for your reply. It's so informative. Thanks again.
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