NPC Neurosphere Assay: An In Vitro Assay to Assess the Migratory Response of Neural Precursor Cells from the Neurosphere
Transcrição
To form the neurosphere, pipette 1 milliliter of 100% expansion media into a 35-millimeter dish without any coating substrate. Then, add one million neural precursor cells in each of these plates and incubate the plate at 37 degrees Celsius for 48 to 96 hours.
To prepare the plate for neurosphere migration, dissolve ECM-mimic gel aliquots in 6 milliliters of 30% expansion medium. Add 1 milliliter of ECM-mimic gel with 30% expansion medium in a single well of a 6-well plate. Then, incubate the plate at 37 degrees Celsius for 30 minutes.
To plate the neurospheres use a pipette to collect the cells in the medium from the 35-millimeter dish and transfer them to a conical tube. Then, spin the tube at 100 x g to pellet the neurospheres. Then, resuspend the pellet in 1 to 3 milliliters of prewarmed 30% expansion medium.
Next, pipette 200 microlitres of the resuspended neurospheres into the 6-well plate containing the ECM-mimic gel with 30% expansion medium and rock the plate. Then, incubate the plate at 37 degrees Celsius for 48 hours. Aspirate the ECM-mimic gel with 30% expansion medium and then fix the cells for 30 minutes using 4% paraformaldehyde.
To analyze the neurospheres, capture images using phase-contrast settings at 10X magnification. Use the NIH ImageJ software to measure the average migration of the neurospheres. On ImageJ, use the freehand line tool to trace the outer contour of the neurosphere.
Then, use measure function to calculate the trace area and trace the inner cell mass of the sphere. Subtract the inner cell mass from the total neurosphere area to quantify the average migration.
