Brain Tumor Stem Cell (BTSC) Migration Assay for Drug Treatment Studies: An In Vitro Live-cell Imaging Technique to Analyze the Effect of Drug Treatment on Migration of BTSCs
Transcript
To assess the effects of a drug treatment of interest on migration, first, pre-treat the cells with the appropriate concentration of the drug for a suitable experimental period. To prepare the chemotaxis plate, coat three membrane insert wells and three bottom reservoir wells per condition of a 96-well chemotaxis plate with 75 and 225 microliters of 0.2 milligrams per milliliter type-I collagen, respectively, for 1 hour at 37 degrees Celsius.
At the end of the incubation, aspirate the collagen solution from each well without scratching the collagen coating and wash the wells two times with sterile PBS. After the last wash, remove the insert from the plate and add 225 microliters of growth factor-free media supplemented with 10% FBS to each bottom reservoir well of the chemotaxis plate.
Then, gently return the top insert to the bottom reservoir at an angle to avoid creating bubbles and add 2.5 times 10 to the third dissociated BTSCs in 50 microliters of growth factor-free media to each insert well. It is important to fully dissociate the BTSCs into single cells and to count them carefully before plating to avoid having cell clumps or too many cells in the top insert.
To image BTSC migration along the FBS gradient, place the plate into a live-cell imaging system and set the automated imaging software to acquire microscopic images of the plate every 1 to 2 hours for up to 72 hours. When the image acquisition is complete, select a new processing definition to distinguish the BTSCs from their insert pores on the bottom side of the membrane, modifying the analysis if it does not accurately distinguish the cells from the background membrane as necessary.
To quantify the cells that have migrated through the pores, collect the data as the cell surface area on the bottom side of the membrane for each of the three replicate wells per condition. Then, graph the cell migration as the area of the cells migrated in micrometers squared over time.
