Använda Fluorescensaktiverad cellsortering för att Undersök Cell-Type-Specific Gene Expression i råtthjärnvävnad

Summary

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

Abstract

Hjärnan består av fyra primära celltyper inklusive neuroner, astrocyter, mikroglia och oligodendrocyter. Även om de inte är den vanligaste celltypen i hjärnan, nervceller är den mest studerade av dessa celltyper med tanke på deras direkta roll i att påverka beteenden. Andra celltyper i hjärnan påverkar också neuronal funktion och beteende via signalmolekyler som de producerar. Neuro måste förstå samspelet mellan celltyperna i hjärnan för att bättre förstå hur dessa interaktioner påverkar neural funktion och sjukdom. Hittills är den vanligaste metoden för att analysera protein eller genuttryck utnyttjar homogenisering av hela vävnadsprov, vanligtvis med blod, och utan hänsyn till celltyp. Detta tillvägagångssätt är en informativ strategi för att undersöka generella förändringar i genen eller proteinuttryck som kan påverka nervfunktion och beteende; Men denna analysmetod inte lämpar sig för en ökad förståelse av cell-typ specifikt genuttryck och effekten av cell-till-cellkommunikation på nervfunktionen. Analys av beteende epigenetik har varit ett område av ökande fokus som undersöker hur modifieringar av deoxiribonukleinsyra (DNA) struktur effekter på lång sikt genuttryck och beteende; dock, kan denna information endast vara relevant om analyseras i en celltyp specifikt sätt med tanke på differential härstamning och därmed epigenetiska markörer som kan förekomma på vissa gener enskilda neurala celltyper. Den fluorescensaktiverad cellsortering (FACS) teknik som beskrivs nedan tillhandahåller ett enkelt och effektivt sätt att isolera enskilda neurala celler för efterföljande analys av genexpression, proteinuttryck, eller epigenetiska modifieringar av DNA. Denna teknik kan även modifieras för att isolera mer specifika neurala celltyper i hjärnan för efterföljande celltyp-specifik analys.

Introduction

The purpose of the protocol described below is to isolate individual cell types from a heterogeneous population of neural tissue for the subsequent analysis of cell-type-specific gene expression, protein expression, or even epigenetic markers. The brain is comprised of many cell types that are derived from distinct progenitor cells and that have cell-type-specific properties and functions. Despite these differences, these distinct neural cell types can express similar receptors and intracellular signaling molecules which make the analysis of these more ubiquitous proteins difficult to measure or interpret in a cell-type-specific manner using conventional methods. Neuroscientists must identify the function and activation of these distinct cell types in the brain and how they can individually impact behavior as this will ultimately be the first step to identifying more specific drugs and therapeutic targets for neurological and neuropsychiatric diseases. Despite this overarching goal of neuroscience research, it can been difficult to isolate individual neural cell types from the brain for the analysis of gene or protein expression, the activation of signaling molecules, or the modification of epigenetic markers on DNA. Of the techniques that are currently used, immunohistochemistry can identify the expression of proteins in a cell-type-specific manner when combined with additional staining of a cell-type-specific marker, though this relies on specific antibodies that can properly stain for the proteins of interest and it can be difficult to quantify. In situ hybridization can identify the specific localization of messenger ribonucleic acid (mRNA) in individual cells in the brain, but this is a laborious process that also limits the co-analysis of specific cell types and only allows for the analysis of one or maybe a few genes of interest. Laser capture micro-dissection uses a laser to isolate subpopulations of cells that are visualized via microscopy; however the time-consuming nature of this process and the relatively low yield can significantly limit the subsequent analysis of proteins or mRNA levels, particularly if the expression of these molecules is low to begin with. Fluorescence activated cell sorting (FACS) is a relatively novel technique in the field of neuroscience to isolate individual cell types from the brain for subsequent analysis of gene expression¹ and/or epigenetic targets². This process can also be used to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, protein expression, or epigenetic markers. FACS has been used in a number of medical research fields such as cancer and immunology for decades to count and sort different cells based on either physical or biochemical characteristics³. In addition, flow cytometry has classically been used to analyze protein expression on a per cell basis, using specific antibodies. The procedure described below, takes advantage of classical flow cytometry techniques to isolate individual cell types for subsequent analysis of molecular biology endpoints. The flow cytometer can analyze several thousand cells in a second, which makes it a quick and efficient alternative to the techniques described above. In addition, cells can be isolated based on the cellular expression of a specific protein (for example a neurotransmitter receptor) or a combination of two or more proteins (colocalization of multiple proteins in a specific cell type). This allows the user to isolate very selective neural cell types based on their molecular properties to identify their function in the brain.

To perform FACS, neural cells are prepared into a single-cell suspension which is passed through a flow cell that carries and aligns the cells so that they pass single-file through a light beam and lasers for analysis. A computer acquires the data from each cell and plots it on a histogram for analysis of specified parameters (size, granularity, and fluorescence). Based on these parameters, the cells can immediately be sorted into separate tubes for their recollection and subsequent analysis of any endpoint desired. The protocol described below utilizes three antibodies to sort neurons (using a Thymocyte antigen 1, Thy1 antibody), astrocytes (using a glial glutamate transporter, GLT1 antibody), and microglia (using a cluster of differentiation molecule 11B, CD11b antibody). This protocol can be used as described below or modified with different antibodies depending on the cell type that one would like to isolate for his own experiments.

There are a few caveats to consider when determining whether this protocol is appropriate for specific experiments. One major caveat may concern the specific cell type that one would like to isolate. In this protocol, the three antibodies that are used are extracellular antibodies, which allow the experimenter to keep the cell types intact during the staining procedure, thus preserving the integrity of the RNA, DNA and proteins inside. It is possible that one may wish to isolate a specific neural cell type using an antibody that identifies a protein that is only expressed inside that particular cell. For example, one might want to isolate dopaminergic neurons using an antibody to tyrosine hydroxylase or isolate acetylcholine neurons using an antibody for choline acetyltransferase. These proteins are intracellular and thus would require fixation and permeabilization of the cell membrane for subsequent staining with the appropriate antibodies. While this has been done before ^4,5, this process may significantly decrease the yield of RNA or DNA from these permeabilized cells. Another caveat may be that not all antibodies are appropriate for FACS. For example, one may currently use an antibody that works very well for western blot, a technique that requires the denaturation of proteins. This antibody may not necessarily be suitable for identification of these cell types using FACS given that the proteins are not denatured at any point in this protocol and thus the antibody may have no way to bind to its inherent antigen. Companies provide specification sheets which identify the applications for which an antibody has been approved. If an antibody has not been approved for flow cytometry, it should not discourage one from trying this protocol with a particular antibody; however, one should be aware that it isn’t guaranteed to work for FACS. A third caveat of this protocol has to do with the number of cells that one is trying to isolate. FACS is an excellent technique to yield the most cells possible from even a small piece of tissue, but it is also possible that if one would like to isolate a relatively sparse population of cells from a relatively small brain region, the yield from one animal will be inherently low. In this case, it may be necessary to pool the brain tissue from a few animals in the same treatment group in order to yield the number of cells necessary for subsequent analysis; however, a recent publication has used gene-targeted pre-amplification of cDNA for subsequent analysis of gene expression from a small number of activated neurons (5-6%) from a larger set sorted neurons, indicating that it is possible to analyze gene expression from even small subsets of neural cells without pooling large numbers of animals⁶A final caveat of this technique is that one should have access to a cell sorter that is not too far away. The cell sorter is a complicated machine that requires significant training in order to use it properly. Thus these machines are often run by a qualified technician in a core facility. In addition, the goal of this procedure is to dissociate the neural tissue, stain it for specific antibodies, and immediately take those samples to a sorter within a short period of time (perhaps a half day). This timeframe will help to increase the survival and yield of isolated cells and maintain the integrity of the cells for subsequent processing and analysis. If all of these parameters described above are met, FACS is an excellent method to analyze cell-type specific expression of genes and proteins from neural tissue.

All experiments were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Protocol

1. Preparation for Tissue Collection (15 – 30 min) Fire polish three sets of glass Pasteur pipettes in decreasing diameters and number them “1” = approximately 1 mm diameter, “2” = approximately 0.75 mm diameter, and “3” = approximately 0.50 mm diameter. Resuspend lyophilized Enzyme A with all of Buffer A according to the protocol provided with the Neural Dissociation Kit (see Table of Materials). Do not vortex. Aliquot the solution into 50 µl aliq…

Representative Results

The Importance of Myelin Depletion and Tissue Perfusion Figure 1 depicts the importance of myelin depletion. Myelin depletion (Steps 5.1 through 5.12 of the protocol above) occurs when the single-cell suspension is incubated in the Myelin Removal Beads, washed, and subsequently passed through the column on the magnetic sorter. The purpose of these steps is to reduce the amount of cellular debris present in each sample. As neurons are dissociated and triturated, it can shear of…

Discussion

Similar to many other tissues and systems, the brain is comprised of a heterogeneous cell population that functions together to impact behavior. The analysis of gene expression, protein expression, or epigenetic modifications from individual cells within that heterogeneous population has the potential to reveal information about the function of the system as a whole, to identify cellular processes regulating both normal behavior and disease processes, and to provide cell-type-specific targets for potential therapies. It …

Materials

Neural Dissociation Kit (P)	Miltenyi Biotec	130-092-628
Myelin Removal Beads II	Miltenyi Biotec	130-096-733
LS Columns	Miltenyi Biotec	130-042-401
QuadroMACS Separator	Miltenyi Biotec	130-090-976
MACS MultiStand	Miltenyi Biotec	130-042-303
Nylon Mesh Sheet	Amazon	CMN-0074-10YD	40 inch width, 80 micron size mesh
Fc Block / anti-CD32	BD Biosciences	BDB550270	reactivity for rat
APC-conjugated CD11b antibody	Biolegend	201809	reactivity for rat
Rabbit anti-GLT1	Novus Biologicals	NBP1-20136	reactivity for rat or human
PE-conjugated anti-rabbit secondary antibody	eBioscience	1037259	secondary antibody for anti-GLT1
FITC-conjugated anti-rat CD90 (Thy1) mouse antibody	Biolegend	202504	reactivity for rat