This protocol uses flow cytometry to quantitatively assess extracellular MHC class 1 expression on primary neurons cultured from mice. NC2 immunostaining for MHC1 expression can also be performed to avoid a loss of signal due to protein, tertiary, structural changes, permeabilization or denaturing conditions. In addition to directing immune responses to infections, MHC class 1 modulates neuronal synaptic connections.
However, the factors that regulate MHC class 1 expression are still unknown. The embryonic brain dissection steps take practice to master. When learning the technique, take care to practice the dissection without worrying about the time or subsequent culturing process.
For isolation of the embryonic hippocampus, place the first harvested mouse embryonic brain under a stereo-dissection microscope. And use two pairs of Sterile Dumont number 5 forceps to pinch off the olfactory bulbs and to thoroughly pull away the meninges. Once the meninges have been completely removed, the superior side of the cortex will open laterally to expose the hippocampus.
Use the forceps to pinch the hippocampus away from the attached cortex and carefully transfer the isolated hippocampus to a Sterile 15 milliliter conical tube containing five milliliters of Hibernate-E Medium on ice. When all of the hippocampi have been collected into a single 15-milliliter tube, sediment the brain tissue by centrifugation. Replace the supernatant with 0.5 milliliters of freshly prepared papain dissociation per embryo and mix the tube several times by inversion.
Place the tissue at 37 degrees Celsius for 30 minutes, mixing the samples by inversion every 10 minutes before collecting the tissue again by centrifugation. Replace the supernatant with an equal volume of fresh Hibernate-E Medium and use a fully open glass, fire polished Pasteur pipette to triturate the tissue 10 times. After letting the tissue settle for two minutes, transfer the supernatant into a new 50-milliliter conical tube.
Add an equal volume of Hibernate-E Medium back to the tissue and use a half open glass fire-polished Pasteur pipette to triturate the tissue 10 times. After letting the tissue settle for another two minutes, pool the supernatant in the 50-milliliter tube. Add an equal volume of Hibernate-E Medium to the tissues and triturate the tissue 10 times with a quarter open glass fire polished Pasteur pipette.
After letting the tissue settle for two minutes, pool the supernatant in the 50-milliliter tube. Collect the dissociated cells in the supernatant by centrifugation. And resuspend the pellet in five milliliters of neuron growth medium for counting Dilute the cells to a final plating density of five times 10 to the fifth viable cells per milliliter of neuron growth medium and add one milliliter of cells to each well of a 12-well poly-D-lysine coated plate.
Then place the plate in the cell culture incubator, replacing half of the medium with an equal volume of fresh medium twice a week for the lifespan of the culture. To assess the ability of the cultured neurons to express MHC1, at the appropriate day of culture, replace 0.5 milliliters of supernatant in each well with 0.5 milliliters of fresh neuron growth medium supplemented with 200 units per milliliter of interferon beta for a six to 72-hour incubation in the cell culture incubator. At the end of the incubation, wash each well one time with cold Neurobasal Medium without supplements before adding 0.5 milliliters of non-supplemented cold Neurobasal Media supplemented with one microgram per milliliter of FC block and one microgram per milliliter of fluorescence conjugated anti-MHC1 antibody to each well.
After a 45-minute incubation at four degrees Celsius, protect it from light. Wash each well one time with cold Dulbeccos PBS. Next, add 0.5 milliliters of room temperature enzyme-free cell dissociation buffer to each well and agitate to dislodge the cells.
Confirm dissociation under an inverted tissue culture microscope and add 0.5 milliliters of FACS buffer to each well. Triturate the cells to disperse clumps and transfer the entire volume from each well into individual 1.7 milliliter microcentrifuge tubes. Collect the cells by centrifugation and resuspend the pellet in 100 microliters of fresh FACS buffer per tube.
Transfer each suspension into individual wells of a 96-well U-bottom plate and add 100 microliters of fixative reagent to each well. Triturate several times to avoid cell clumping and incubate the plate for 15 minutes at room temperature protected from light. At the end of the incubation, centrifuge to collect the cells at the bottom of the wells and resuspend the pellets in 200 microliters of fresh FACS buffer per well.
After centrifuging, resuspend the pellets in 100 microliters of permeabilization reagent supplemented with fluorescence conjugated anti-neuronal nuclei antibody per well. After mixing, incubate the plate for 20 minutes at room temperature with rocking protected from light. At the end of the incubation, collect the cells by centrifugation three times, resuspending the pellets in 100 microliters of fresh FACS buffer between centrifugations.
After the last wash, resuspend the cells in 100 microliters of 2%pair of formaldehyde in FACS buffer with thorough mixing. Neurons can be identified through the sequential gating of total events to exclude cellular debris and doublets and by their neuronal nuclei positivity. Neuronal nuclei positive cells can then be further analyzed for their MHC1 positivity.
From this data, the percentage of neurons positive for MHC1 staining and the median fluorescence intensity can be calculated, revealing that, for example, interferon beta treatment significantly upregulates the percentage and intensity of MHC1 neuron expression. With slight modifications, these methods can be used to culture other neuronal populations like cortical neurons or to test different cellular markers, stimulating molecules, or genetic modifications.
