Melanoma Stem Cell Isolation: A Method to Obtain CD133 Positive and Negative Melanoma Cells Using Magnetic-activated Cell Sorting
Trascrizione
– Melanoma harbors a small subpopulation of cancer stem cells possessing both self-renewal and tumor initiation capabilities. CD133 or Prominin-1, a pentaspan transmembrane glycoprotein, is one of the key markers expressed by metastatic melanoma cancer stem cells. To isolate CD133-positive cells, begin with a suspension of primary melanoma cells in an appropriate buffer.
Incubate with a blocking reagent containing proteins that block non-specific binding sites on the cell surface. Treat the cells with biotinylated CD133 primary antibodies. These antibodies bind to CD133 antigens expressed on CD133-positive cells. Now, add anti-biotin magnetic microbeads to the suspension. Incubate for the microbeads to bind to the biotin-labeled cells.
Finally, pass the incubated suspension through a column placed in a magnetic field. With magnetic influence, all CD133-positive cells labeled with the microbeads stick to the column, while the unlabeled cells pass through. Collect these unlabeled CD133-negative cells. Remove the column from the magnetic field. Flush the contents using a suitable buffer to elute and collect CD133-positive cells.
In the following protocol, we will show the isolation of CD133-positive and negative cancer stem cells from a culture of primary melanoma obtained from tumor tissue. Wash 80% confluent cells with pre-warmed PBS. Add appropriate amount of Accutase and incubate until cells detach from the culture surface. Harvest the cells in Quantum 263 medium and transfer to a 50 milliliter tube.
Enumerate the cells. Then, centrifuge the required number of cells for 5 minutes at 300 x g and 4 to 8 degrees Celsius. Aspirate supernatant completely, and resuspend up to 1 times 10 to the eighth cells in 350 microliters MACS buffer. Add 100 microliters FcR Blocking Reagent and 50 microliters CD133/1-Biotin. Mix well and incubate at 4 degrees Celsius for 10 minutes.
Wash the cells with 10 milliliters MACS buffer, followed by centrifugation for 5 minutes at 300 x g and 4 to 8 degrees Celsius. Aspirate the supernatant completely. After a second wash, resuspend the cell pellet in 400 microliters of MACS buffer. Add 100 microliters anti-biotin microbeads and mix well. Incubate at 4 to 8 degrees Celsius for 15 minutes.
Meanwhile, to prepare the MACS separator for the magnetic separation, attach the QuadroMACS to the MACS Separator Multi Stand. Insert the required number of LS Columns with the column wings to the front in the magnetic field of the QuadroMACS. Place a Pre-Separation Filter into each LS Column and an appropriate collection tube under each column.
Rinse the filter and column with 3 milliliters MACS buffer and discard the flow through. After washing the cells in MACS buffer as described earlier, resuspend the cells in 500 microliters MACS buffer and apply the cell suspension onto the prepared LS Column. Wash three times with 3 milliliters MACS buffer per column. Collect the total effluent of the unlabeled CD133-negative cell fraction.
Next, discard the Pre-Separation Filter, remove the column from the separator and place it on a suitable collection tube. Apply 5 milliliters MACS buffer. Immediately flush out the labeled CD133-positive cells by firmly pushing the plunger into the column. To increase the purity of the negative fraction, apply the cell suspension onto a new equilibrated LS Column and collect the effluent CD133-negative fraction.
