Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The overall goal of this procedure is to isolate individual neural cell types from brain tissue for subsequent analysis. This is accomplished by first dissociating neural tissue into a single cell suspension. In the second step, the myelin is removed from the suspension and then the cells are stained for cell type specific antibodies.

In the final step, the individual cell populations are sorted by flow cytometry. Ultimately, this method of cell type specific isolation will allow users to assay changes in gene or protein expression or epigenetic modifications in a cell type specific manner. This method can help answer key questions in the field of neuroscience, such as which cell types express certain receptors, which express certain proteins, and which express certain epigenetic modifications so that we can better understand cell type specific mechanisms in the brain.

Begin the neural dissociation by using sterile razor blades to dice each sample harvested from adult brain tissues into small 30 to 400 milligram pieces on the lid of a six Well culture plate. Then immerse the tissue in one milliliter of calcium and magnesium free HBSS and use a pipette to transfer the samples into sterile two milliliter centrifuge tubes when all of the tissues have been collected. Centrifuge the samples and aspirate the supernatants.

Add 1, 900 microliters of warm enzyme. Mix one and incubate the samples for 15 minutes. In a 37 degree Celsius water bath, inverting the tube several times every five minutes to resuspend the settled pieces of tissue.

At the end of the incubation, add 30 microliters of freshly prepared enzyme. Mix two to each sample and gently invert the tubes. Then using a pest pipette labeled one, dissociate each sample with 30 up and down iterations.

Taking care to avoid generating bubbles. Incubate the samples for another 15 minutes at 37 degrees Celsius with inversion every five minutes, followed by dissociation of the samples with pest pipette. Number two, as just demonstrated.

Then switch to pipette Number three, repeat the dissociation and incubate the samples for 10 more minutes. In the water bath with inversion every five minutes at the end of the incubation filter the single cell suspensions through 80 micron cell strainers into 15 milliliter Falcon tubes. Washing each strainer with 10 milliliters of calcium and magnesium free HBSS to stop the enzymatic reactions when the last sample has been filtered, spin down the tubes and aspirate the supernatants to deplete the myelin from the cell.

Suspensions thoroughly resuspend the pellets in 400 microliters of myelin removal buffer, and add the appropriate amount of myelin removal beads to each tube. Pipette the solutions up and down until the cells and beads are thoroughly mixed. Then incubate the sample at four degrees Celsius for 15 minutes.

After the incubation wash each sample in five milliliters of myelin removal buffer while the cells are spinning, place one column for each sample in the magnetic field of a magnetic sorter and place a clean 80 micron filter on top of each column. Rinse each filter and column with one milliliter of myelin removal buffer three times collecting all of the flow through in a waste container. After the final rinse, place five milliliter polystyrene round bottom tubes directly beneath each column to collect the eluate.

Then aspirate the super named from the samples and resuspend the pellets in 500 microliters of myelin removal buffer. Immediately apply the cell suspensions to the columns, collecting the cells in the tubes below. Then rinse each filter and column four times with one milliliter of myelin removal, buffer per wash.

After the last wash, spin down the cells and aspirate the supernatants to stain the cells for sorting by flow cytometry. Vortex each sample for about two seconds to dissociate the pellets and transfer one microliter of cells from each tube into individual tubes. For the staining controls, immediately add five microliters of FC block to each tube, including the staining controls.

Then vortex the samples again and incubate them at four degrees Celsius. After five minutes, add 100 microliters of the appropriate primary antibody cocktail. Cover the tubes to protect the antibodies from light and incubate the cells at four degrees Celsius for 20 minutes.

At the end of the incubation, wash the samples in two milliliters of wash buffer, then aspirate the supernatants and label each sample with 100 microliters of the appropriate secondary antibody. Cover the tubes and place them at four degrees Celsius for 15 minutes. Then wash the samples in two milliliters of wash buffer centrifuge the samples, and then aspirate off.

Supernatants Resus, suspend the pellets in 250 microliters of sterile PBS and sort the cells here representative flow sorted populations of neural cells from a single male hippocampus are shown the cells were gated first from all possible events based on their forward and side scatter properties. Next, the single cells or singlets were gated from any doublets or larger clumps of cells based on their size to allow accurate sorting of the individual cell types, the single cells were then sorted into CD 11, B positive, and CD 11 B negative cell populations with further sorting of the CD 11 B negative cells into GT one and TH one positive cells. The analysis program was then used to determine the total number of events in each population as well as the percentage of the parent populations to confirm the purity of the sorted cells.

In this particular example, the relative gene expression of alternative cell type specific genes was analyzed by real-time PCR. As expected, these data confirmed the purity of the sorted cells, as well as identified some interesting differences across the investigated brain regions. Following confirmation of the purity of the sort, the expression of other genes of interest can be measured to determine the specific cell type in which they are expressed, or whether their expression is altered following treatment in a cell type specific manner.

For example, here the expression of cow binding, a calcium binding protein often used to identify neurons in the hippocampus and the cerebellum was investigated. After watching this video, you should have a good understanding of how to isolate specific neural cell types of interests, such as the neural cell shown here using appropriate cell surface antibodies.