Hi all,
I would like to do Dio staining in non-differentiate and differentiae SHSY5Y cells.
I really appreciate if you could share Dio staining protocol with me.
What is the purpose of the staining with DiO? Cell tracking (and for how long)? Membrane localization? Cell-cell fusion visualization?
Of course, I can provide you with a basic protocol for Dio (3,3'-Dihexadecyloxacarbocyanine perchlorate) staining in mesenchymal cells, both undifferentiated and differentiated. Dio is a lipophilic fluorescent marker used to label cell membranes. Here is the protocol: For your convenience I remove the mesenchymal part and put the acronym that you put for your cells. Materials: Dio solution (you can purchase it commercially). Undifferentiated or differentiated SH-SY5Y cells. Suitable culture medium. Centrifuge tubes. Fluorescence microscope. Procedure: Cell culture: Culture your undifferentiated or differentiated SH-SY5Y cells in culture dishes with the appropriate culture medium. Make sure the cells are in optimal condition before staining. Preparation of Dio solution: Prepare a 1 μg/mL solution of Dio in culture medium without fetal bovine serum (FBS). Dio is sensitive to light, so keep the solution in the dark. Staining: Remove the culture medium from the cells and wash them with PBS (phosphate-buffered saline) once or twice. Add the Dio solution (1 μg/mL) to the culture medium without FBS and place it on the cells. Incubate cells for 15-30 minutes at 37°C in a CO2 incubator to allow Dio to penetrate cell membranes. Washed: Wash the cells twice with PBS to remove excess Dio. Mounting: You can mount the cells on a glass slide with a mounting medium suitable for fluorescence microscopy. This will allow observation under the microscope. Observation: Use a fluorescence microscope with appropriate excitation (usually around 488 nm) and an emission filter in the range of 500-550 nm to observe Dio fluorescence. You should be able to see the cell membrane of the labeled cells.
My purpose is to labeling dendritic spines in cell cultures to study morphology of my neuronal cells
Thank you so much dear Alberto Daniel Saucedo-Campos for your reply
Then I agree with Alberto's protocol as a good starting point, though you may need to optimize the time and concentration.
Hi Jason:
The staining with DiO (Dialkylcarbocyanine, also known as 3,3'-dioctadecyloxacarbocyanine perchlorate) is a commonly used technique in cell and molecular biology with several purposes, and some of the ones you mentioned are valid applications. Here are the main purposes of DiO staining:
The duration of cell tracking with DiO staining can vary depending on your experimental needs. It can be used for short-term studies involving tracking for a few hours or days, or it can also be used for long-term experiments involving tracking for weeks or more, depending on the viability of the labeled cells and the culture conditions.
In summary, DiO staining is a versatile tool used for various purposes in cell and molecular biology, and the tracking duration depends on the specific objectives of your study.
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