Fluorescent Labeling of Glioma Cells: A Lentiviral Vector-based Transfection Method to Obtain Glioma Cells Expressing Fluorescent Protein for Deep Tissue Imaging
DEŞİFRE METNİ
In vivo brain cancer imaging involves injecting fluorescently-labeled cancer cells into the mouse brain to observe tumor development. For fluorescent labeling, incubate glioma cells with lentivirus particles. The lentiviral vector carries the gene for infrared fluorescent protein, iRFP.
The virus binds to the glioma cell surface and releases RNA into the cell. Cellular enzymes convert the viral RNA to double-stranded DNA, which integrates into the cellular genome and allows iRFP expression. iRFP's excitation and emission maxima lie within a near-infrared region where tissues have minimal absorbance and scattering. This allows accurate deep tissue imaging.
Next, remove the spent medium and add trypsin. Trypsin, a protease enzyme, digests the adhesion proteins, detaching the cells from the flask surface. Pipette repeatedly to separate the individual cells. Collect the cell suspension into a tube and centrifuge. Now, add a suitable buffer to resuspend the pellet.
Dispense the cell suspension into flow cytometry tubes and stain the cells with a fluorescent dye, DAPI. DAPI stains the nucleus of dead cells bright blue, distinguishing them from live cells. Using a fluorescence-activated cell sorter or FACS, sort the live iRFP positive cells from other cell populations based on their fluorescence. The isolated cells can be used for injection into mice for tumor imaging.
