Fluorescent Labeling of Glioma Cells: A Lentiviral Vector-based Transfection Method to Obtain Glioma Cells Expressing Fluorescent Protein for Deep Tissue Imaging
筆記録
Begin by seeding 1 times 10 to the sixth glioblastoma cells in 5 milliliters of medium into a 10-centimeter dish for a 24-hour incubation in a cell culture incubator. The next morning, transduce the cells with lentivirus expressing an appropriate fluorescent protein of interest at a multiplicity of infection of 5 and return the cells to the incubator.
After 24 hours, replace the supernatant with 5 milliliters of fresh medium and incubate the culture for another 48 hours. At the end of the incubation, replace the medium with 3 to 5 milliliters of trypsin for 10 to 15 minutes at 37 degrees Celsius, followed by careful pipetting to fully dissociate the detached cells.
Collect the cells by centrifugation and resuspend the pellet in 500 microliters of sorting solution. Split the cells into the appropriate number of FACS tubes and costain the cells with DAPI for dead cell exclusion. Then, load the tubes onto the flow cytometer and sort the live cells according to their fluorescent construct expression into a 15-milliliter conical tube containing fresh sorting solution.
After centrifugation, resuspend the construct-positive/DAPI-negative cells in 5 milliliters of culture medium and seed them onto a new 10-centimeter dish for their culture at 37 degrees Celsius for 48 to 72 hours. At the end of the incubation, split the cells for seeding into multiple dishes for subsequent in vivo experiments.
