The overall goal of this technique is to use immunomagnetic isolation to select O4 positive oligodendrocytes from neonatal mice pups for their in vitro culture analysis. This method can help answer key questions in neural science field related to the study of diseases that affect myelin and myelination. The main advantage of this technique is that it facilitates the preparation of final oligodendrocyte culture of a greater than 80%purity in about hour hours.
Begin by using small dissecting scissors to cut the skin along the midline of the scalp of a day five to seven postnatal day mouse. Retract the skin flap to expose the skull, and cut the skull carefully along the midline from the opening in the back to the frontal area. From the opening in the back of the skull, cut toward each eye socket along the base of the bone and use fine forceps to gently tease the cortices away from the midbrain.
Then, transfer the cortices to a 60 millimeter tissue culture dish containing seven milliliters of B-27 Neurobasal A Medium. When all of the brains have been collected, transfer the cortices into a new 60 millimeter tissue culture dish containing five milliliters of dissociation buffer, and use a number 15 scalpel blade to dice the cortices into one cubed millimeter pieces. Incubate the brain pieces in a 37 degree Celsius 5%CO2 incubator for 20 minutes.
Then, add one milliliter of Bovine Growth Serum to stop the enzymatic reaction. Using a 10 milliliter Serological Pipette, transfer the tissue slurry into a 15 milliliter conical tube and gently dissociate the brain tissue six to eight times with pipetting. Allow the dissociated tissue chunks to settle for two to three minutes.
Then, transfer the supernatant into a fresh tube. Next, add three milliliters of Neurobasal A Medium, supplemented with Bovine Growth Serum and DNase I to the tissues, and use a five milliliter pipette to gently dissociate the tissues with more pipetting. You see, the appropriate pipetting force is crucial to ensure a high yield of viable cells.
If pipetting is too hard, the viability might be low. Whereas if pipetting is too gentle, the cellular yield might be low. Allow the tissue pieces to settle for another two to three minutes.
Then, transfer the supernatant into a new tube, and add three milliliters of fresh Neurobasal A Medium, supplemented with Bovine Growth Serum and DNase I to the tissue. Gently dissociate the brain tissue with a P-1000 pipette tip until no large chunk of tissue remain, taking care to avoid bubbles. Use a 10 milliliter pipette to filter the cell solution through a 70 micron cell strainer into a 50 milliliter conical tube, and bring the final volume of the single-cell suspension to 30 milliliters with fresh Neurobasal A Medium, supplemented with Bovine Growth Serum and DNase I.Split the cell suspension to two 15 milliliter conical tubes, and pellet the neural cells by centrifugation.
Remove all but the last five to 10 microliters of cloudy supernatant and add three milliliters of Neurobasal A Medium supplemented with Bovine Growth Serum to the cells. Carefully re-suspend the cells pellet and bring the final volume up to 15 milliliters with fresh Neurobasal A Medium containing 10%Bovine Growth Serum. Filter the cell solution through a 40 micron cell strainer into a new 50 milliliter conical tube, and bring the final volume up to 30 milliliters with fresh Neurobasal A Medium containing 10%Bovine Growth Serum.
Then, split the filtered cell suspension between two 15 milliliter conical tubes for centrifugation. and re-suspend the pellets in 2.5 milliliters of ice cold Magnetic Cell Sorting Buffer before pooling the cells. After counting, centrifuge the cells again, and re-suspend the pellet in 90 microliters of fresh Magnetic Cell Sorting Buffer per one times 10 to the seven cells.
Next, add 10 microliters of anti-04 beads per one times 10 to the seven cells, and mix the cell solution with gentle flicking. After 15 minutes at four degrees Celsius with a gentle flicking every five minutes, gently add two milliliters of Magnetic Cell Sorting Buffer per one times 10 to the seven cells for a wash by centrifugation, and discard the supernatant by careful vacuum aspiration. Re-suspend the pellet in 500 microliters of fresh Magnetic Cell Sorting Buffer for every one times 10 to the seven cells, and place an appropriately sized magnetic beads sorting column into its corresponding magnetic separator.
Place a 40 micron strainer onto the column and pre-rinse the strainer with three milliliters of Magnetic Cell Sorting Buffer, letting the buffer run through the column without letting the column dry. Add the cells to the strainer, followed by a one milliliter wash with Magnetic Cell Sorting Buffer. Wash the column three times with three milliliters of Magnetic Cell Sorting Buffer per wash, and one time with Oligodendrocyte Proliferation Medium.
Then, transfer the column into a 15 milliliter tube, and use the plunger to immediately flush five milliliters of fresh Oligodendrocyte Proliferation Medium through the column. After counting, dilute the cells to an appropriate plating density, and replace the laminin from pre-coated cover slips in a 24 well plate with 100 microliters of Oligodendrocyte Proliferation Medium. Incubate the oligodendrocytes at 37 degrees Celsius and 5%CO2 for no longer than 45 minutes to promote oligodendrocyte adhesion to the cover slips.
Then, flood each well of the 24 well plate with 500 microliters of Oligodendrocyte Proliferation Medium for a 24 hour incubation. The next day, replace the supernatants with Oligodendrocyte Differentiation Medium, and return the cells to the incubator until they are ready for fixation. 24 hours after plating, the cells appear to be bi or tripolar under Phase Contrast Microscopy, a characteristic feature of early stage proliferating oligodendrocyte.
Immunofluorescence Staining shows that 24 hours after plating, the majority of these pre-oligodendrocytes also demonstrate NG2 and O4 labeling, while labeling with GFAP and anti-01 antibody are much less common, suggesting that the majority of cells at this stage ae pre-oligodendrocytes with few immature or mature oligodendrocytes. At 72 hours, the oligodendrocyte morphology appears to be more complex than at 24 hours, and there is a much lower frequency of cells positive for the early oligodendrocyte marker, NG2. Also, most cells demonstrate O4 labeling, and almost half of the cells stained for 01 antibody at this stage, suggesting that the cells are differentiating into a more mature phenotype.
Notably, the presence of astrocytes remains extremely low. These results indicate that over time, immuno-magnetically isolated oligodendrocytes are able to differentiate in vitro into Stage Three of maturation with very little contamination of other cells types, such as astrocytes. After watching this video, you should have a good understanding of how to isolate primary oligodendrocytes from neonatal mouse pups using immunomagnetic isolation.
Once mastered, this technique can be completed in four hours, if it is performed properly. Thanks for watching. Good luck with your experiments.
