This article describes a methodology for studying glioma cell migration using a neurosphere model. The approach utilizes live-cell imaging to observe the behavior of glioma cells in a tumor-like microenvironment.
Begin with a flat-bottom multi-well plate coated with a basement membrane matrix, which contains extracellular polymer and adhesion proteins that mimic the tumor microenvironment's matrix.
Introduce a media containing a neurosphere of glioma cells derived from a pediatric brain tumor, ensuring it is centrally positioned in the well.
The matrix promotes the neurosphere attachment to the well.
Add a background suppressor reagent to minimize non-specific fluorescence.
Next, introduce green fluorophore-conjugated anti-CD44 antibodies.
Transfer the plate to a live-cell imaging system housed within an incubator that maintains the cell's physiological conditions.
Over time, glioma cells express the cell-adhesion protein CD44, which interacts with the adhesion proteins.
This activates the cell signaling pathway and facilitates cell migration from the neurosphere.
The fluorophore-conjugated anti-CD44 antibodies bind to the expressed CD44 on the glioma cells, making them fluoresce.
The appearance of green fluorescent cells moving away from the neurosphere confirms the glioma cell migration.
Once the wells are coated with BMM, cut a P200 tip. Take 50 microliters of the cell medium and NS from each selected well, and transfer it to a coated, flat, bottom well. Check the presence and the position of the NS in each well visually. Gently add 50 microliters of the diluted BSR and ALR antibody to each well, waiting for two to three minutes to let the reagents mix. And using an inverted microscope, ensure that most of the replicate NS are centrally located in the well. After ensuring the absence of any bubble, gently transfer the plate in the live cell analysis instrument as demonstrated previously.
On the live cell analysis instrument software, select the option Schedule To Acquire. Then, click on the plus tab and select Scan On Schedule to scan the plates with intervals as mentioned in the text manuscript. On the software window, create or restore vessel, and then click on the option New. Select Dilution Cloning scan type, 4X objective, and phase and green for the migration assay. Select the plate type and define the wells to be scanned by highlighting them on the plate map. Then, set up the scanning frequency. Click on Add To Schedule and start the scan.
繁體中文
