The development of selective gene and drug delivery tools to treat the neurodegenerative disorders, requires the quantification and characterization of cell targeting, upon re-administration, or viral vectors, or nanoparticles. They have throughput and multiplexing capabilities of flow cytometry, allows the straightforward and simultaneous identification of multiple cell types from the mouse brain and spinal cord. Demonstrating the procedure will be Francisco Javier Molina Estevez, a post-doc from Alexander's Laboratory.
After harvesting the brain and spinal cord from an eight-week-old C57 Black 6 mouse, place the tissues into individual wells of a six-well plate, containing two milliliters of ice cold HPSS per well on ice. Divide each harvested tissue into two equal pieces. And use scissors to mince one half of each tissue sample into one to two millimeter thick pieces.
When all of the tissues have been fragmented, pre-rinse a modified 1, 000 microliter pipette tip in HPSS, and use the pipette to transfer each tissue suspension into individual 15 milliliter conical tubes. Rinse the wells with an additional two milliliters of HPSS per well. And pull the washes in the corresponding 15 milliliter conical tubes.
Sediment the samples by centrifugation. And mix 50 microliters of Enzyme P from a neural tissue dissociation kit with 1, 900 microliters of Buffer X from the kit per sample. Warm the enzyme mix at 37 degrees celsius for at least 10 minutes.
And aspirate the supernatant from each tissue collection tube. When the enzyme mixture is ready, add 1.95 milliliters of the solution to each sample. And gently vortex to re-suspend the pellets.
Next, incubate the samples on a wheel for 15 minutes at 37 degrees celsius, and mix 10 microliters of Enzyme A with 20 microliters of Buffer Y per sample. Prewarm the second enzyme solution at 37 degrees celsius, before adding 30 microliters of the solution to each tissue solution at the end of the shaking incubation. Use a 1, 000 microliter pipette tip, pre-rinsed with HPSS to gently mix each sample.
And return the specimens to the wheel for 15 minutes at 37 degrees celsius. At the end of the incubation, arrest the enzymatic reactions with 10 milliliters of ice-cold HPSS. And sediment the samples by centrifugation.
At the end of the centrifugation, re-suspend the pellets in seven milliliters of ice-cold HPSS per tube. And gently vortex each sample, before holding the tubes on ice. For tissue homogenization, add three milliliters of pre-chilled HPSS to the pre-chilled glass mortar of a dounce tissue grinder, and transfer the second half of one of the harvested brain or spinal cord tissue samples to the mortar.
Gently macerate the tissue with 10 strokes of Pestle A, followed by 10 strokes of Pestle B.And transfer the homogenized mixture into a new 15 milliliter conical tube. Fill the tube to a final volume of 10 milliliters with pre-chilled HPSS for centrifugation. And re-suspend the pellet in seven milliliters of fresh HPSS with vortexing, before holding the sample on ice.
For debris removal, add three milliliters of pre-chilled isotonic Percoll solution to each digested or homogenized sample. And gently vortex the samples to make sure they are homogeneously mixed. Next, centrifuge the samples, and carefully remove the whitish disc of debris and myelin floating at the surface of the solution.
When the debris has been discarded, collect all but the last 100 microliters of the supernatant from each tube without disturbing the pellets. Re-suspend each pellet in one milliliter of FACS BL solution for transfer into individual 1.5 milliliter microcentrifuge tubes. After this integration with the Percoll solution, it's important to remove the debris disk and the supernatant very carefully without dislodging the cell pellet to avoid sample loss.
After centrifugation, carefully aspirate the supernatant from each tube, and re-suspend the pellets in 350 microliters of fresh FACS BL per tube. For flow cytometric analysis of the isolated cell types, incubate the samples with five micrograms per milliliter of FC block per tube for 10 minutes at four degrees celsius before adding the appropriate antibody to each tube according to the standing protocol outlined in the table. Vortex each tube for five seconds to mix.
And place the samples at four degrees celsius for 15 minutes protected from light. At the end of the incubation, wash the samples with one milliliter of PBS per tube, and centrifuge. Re-suspend the pellets in the appropriate volume of streptavidin per tube.
After vortexing to mix, incubate the samples for 10 minutes at four degrees celsius protected from light. And wash the cells with one milliliter of PBS per tube as demonstrated. After discarding the supernatants, re-suspend the pellets in 300 microliters of fresh FACS BL per tube.
And label each sample with five microliters of 7-AAD. Then store the samples at four degrees celsius protected from light until cytofluorometric analysis. The homogenization method produces a higher cell yield from both the brain and spinal cord overall, but the majority of the cells retrieved are typically dead, resulting in only and an approximately 14%healed of viable cells from the brain, and an approximately 10%healed from the spinal cord.
In contrast, the papain digestion method results in an overall better preservation of cellular viability. Analysis of the brain and spinal cord cell suspensions with a nine color bi-flow cytometry, reveals the presence of CD45+CD11b+microglia, and macrophages. And CD45+CD11b-lymphocytes in both tissues.
The CD45-cell populations can then be discriminated according to their positivity for astrocyte, or oligodendrocyte markers, or ferional and endothelial surface marker expression. Using the homogenization method, between 32 to 38%of the viable cells are of hematopoietic origin. While the enzymatic digestion method results in the acquisition of a very large fraction of a non-hematopoietic CD45-cells.
Remarkably, CD45+CD11b+microglia and macrophages represent the most abundant viable cell fraction with the homogenization method. The digestion method, however, produces a more heterogeneous representation of cell types including astrocytes, oligodendrocytes, endothelial cells, and neurons. This protocol is versatile, and could be exploited for several downstream applications such as the quantification or isolation of specific cells subpopulations, primary culture, or biochemical, or RNA sequencing analysis.
We have implemented this protocol to assess the gene expression and functional signatures of different CNS populations during this progression, or after treatment in a mouse model of endotrophic lateral sclerosis.
