Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The overall goal of this procedure is to dissociate Fos-expressing neurons from fresh or frozen adult rat brain tissue for subsequent fluorescence activated cell sorting, or FACS. This method can be used to isolate neuronal cells of interest for identify the unique molecular neuroadaptation of behavioral activated neurons. The main advantage of this technique is that it allows the use of frozen tissue, facilitating the isolation of cells from multiple brain areas on different days.

To process this fresh brain tissue, insert two or more prechilled razor blades into the slots of an ice-cold brain slicing matrix to cut one to two millimeter coronal slices containing the brain regions of interest, placing the sections onto a chilled glass plate as they are cut. First, dissect the brain region of interest, discard the other areas, and remove the white matter. To freeze the tissue, transfer the samples into a microtube, and submerge the tube in negative 40 degrees Celsius isopentane for 20 seconds for storage at negative 80 degrees Celsius until further processing.

To mince the tissue, allow the samples to thaw on a cold glass plate for no more than one 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 one 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 single-cell suspension, pellet the samples by centrifugation.

Discard the supernatant. Then, slowly add one 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 four 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 triturate the samples 10 times. Place the sample on ice for two minutes to allow the debris and undissociated cells to settle to the bottom on the tube. Then, transfer the supernatant to a new 15 milliliter tube.

Next, add 600 microliters of Buffer A solution to the remaining pellet, and use a 0.8 millimeter diameter pipette to triturate the sample an additional 10 times. After allowing the tissues to settle as just demonstrated, pool the supernatant with the first cell suspension, and add another 600 microliters of buffer to the pellet. Then, use a 0.4 millimeter pipette to triturate the sample, and place the tube on ice for two minutes to allow the tissues to settle.

Following the mechanical trituration steps in the protocol is important for a successful sorting. Afterwards, pool the supernatant with the dissociated cells. Then, add 600 microliters of Buffer A to the sample, and repeat the 0.4 millimeter pipette trituration three more times.

Place the tubes on ice for two minutes to allow the cells to settle. And then, pool the supernatant in a second 15 milliliter tube. Repeat the trituration process three more times, using the smallest glass pipette, significantly increases the yield of the cells for subsequent sorting.

After all of the supernatants have been collected, add 800 microliters of negative 20 degrees Celsius 100%ethanol to four mirotubes on ice. Then, transfer 800 microliters of cells from the first tube of collected supernatant into each of the first two tubes of ethanol and 800 microliters of cells from the second tube of supernatant into each of the last 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 five 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.

Filter each of the two cell suspensions separately using two different pairs of cell strainers. The cells are then ready for labeling with antibodies against the cell markers of interest and sorting by flow cytometry. Analysis of fresh and frozen brain tissue samples reveals the presence of small homogeneous populations of neuronal cells with a similar size and granularity.

The small homogeneous populations can be gated to exclude any cell aggregates, and the neurons can be identified by their NeuN and DAPI positivity. In both fresh and frozen tissue samples, NeuN mRNA levels are also significantly greater in the NeuN-positive populations than in the NeuN-negative populations. In contrast, the glial and olidogodendrocyte cell marker mRNA levels are greater in the NeuN-negative population than in the NeuN-positive population.

A similar trend of higher microgial cell marker mRNA levels is observed in the NeuN-negative population, although this was not statistically significant. Further, Fos mRNA levels are greater in the Fos-positive neurons than in the Fos-negative neurons, with a three-fold increase of Fos mRNA in the Fos-positive neurons from fresh tissue, and an eleven-fold increase of Fos mRNA in Fos-positive neurons from frozen tissue, confirming the validity of the method for identifying specific genes of interest from flow-sorted cells under both fresh and frozen tissue conditions. While attempting this procedure, it's important to remember to follow the critical cell lysis prevention steps.

Following this procedure, other methods like fluorescence activated cell sorting can be performed to isolate the cell subsets of interest for further downstream molecular analysis. Using frozen tissue allows the samples to be sorted on separate days, critical for experiments that involve lengthy and complicated tissue harvest and preparation procedures. This technique can also be used to prepare postmortem frozen human brain samples stored for several months or years for sorting by flow cytometry.

This technique paved the way for researchers in the neuroscience field for exploring the molecular mechanisms underlying the behavior of Fos-expressing neurons or other immunolabeled wild-type cells of interest. After watching this video, you should have a good understanding of how to dissociate cells from fresh or frozen rat brain tissue for fluorescence activated cell sorting of neurons or cell types of interest.