This article details a protocol for isolating neural cells from rat brain tissue. The process involves mincing the tissue, enzymatic digestion, and cell dissociation to obtain a single-cell suspension for further analysis.
Thaw a section of frozen rat brain tissue.
Add buffer and mince the tissue. Then, transfer it to a tube containing buffer and mix.
Centrifuge the mixture and discard the supernatant.
Add a proteolytic enzyme solution to the tissue and resuspend it using a large-diameter pipette.
Incubate the mixture to break down the extracellular matrix, loosening the neural cells.
Centrifuge the mixture and discard the supernatant.
Resuspend the tissue fragments in a chilled buffer and pipette them repeatedly to dissociate the cells.
Allow the debris and undissociated cells to settle, then transfer the single-cell suspension to a new tube.
Transfer the cells into ice-cold ethanol and mix. Incubate them to fix and permeabilize the cells.
Centrifuge the suspension and discard the supernatant.
Resuspend the neural cells in a cold buffer for further analysis.
To mince the tissue, allow the samples to thaw on a cold glass plate for no more than 1 minute before covering the samples in one to two drops of buffer A solution. Next, holding a razor blade completely vertical to the plate, mince the tissue 100 times in each orthogonal direction. To thoroughly mince the tissue, it is critical to keep the tissue wet with buffer A and follow the specified mincing times and direction.
It is critical to remove the majority of the white matter during the tissue dissection, and to keep the tissue covered in buffer A, to improve mincing efficiency and to maintain the health of the cells.
When the tissue has been thoroughly processed, use the razor blade to transfer the pieces into a microcentrifuge tube containing 1 milliliter of cold buffer A, and invert the tube three to five times to submerge all of the minced tissue in the solution. To dissociate the tissue fragments into a single cell suspension, pellet the samples by centrifugation.
Discard the supernatant, then slowly add 1 milliliter of cold, freshly thawed enzyme solution down the inner wall of the microtube and use a large tip diameter pipette to immediately aspirate and dispense the entirety of each pellet four times to disperse the minced tissue pieces. Next, quickly invert the microtube to prevent the pellets from sticking to the tube bottoms, and incubate the sample with end-over-end mixing for 30 minutes at 4 degrees Celsius.
At the end of the digestion, centrifuge the tube again and add 0.6 milliliters of cold buffer A to the cells. Using the same pipette tip, immediately aspirate and dispense the pellets five times, then select a 1.3 millimeter glass pipette, and use it to gently titrate the samples 10 times.
Place the sample on ice for 2 minutes to allow the debris and undissociated cells to settle to the bottom of the tube. Then, transfer the supernatant to a new 15 milliliter tube. Then, transfer 800 microliters of cells from the first tube of collected supernatant into each of the first two tubes of ethanol.
Invert the tubes to mix the samples. Then place the samples on ice for 15 minutes with mixing by inversion every 5 minutes. At the end of the incubation, centrifuge the cells. Then touching only the wall of the microtube, use a micropipette to slowly remove all but the last 50 microliters of each supernatant.
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