Our research program is funded through the Emmy Noether-Program of the German Research Foundation (DFG), grant PR1132/3-1. Further support by the M&#252;ller-Fahnenberg Foundation of the University of Freiburg is gratefully acknowledged. This study was supported in part by the Excellence Initiative of the German Research Foundation (GSC-4, Spemann Graduate School).

1. Neural Cell Harvesting
<ol>
	<li>Assessment by microscope:
	<ol>
		<li>Prior to initiating an experiment, check the status of the culture with bright-field or phase contrast microscopy. 
		NOTE: While primary neural tissue obtained from dissections is, in principle, equally amenable to flow cytometric analysis14,28, please note that the protocol’s focus is on cells obtained from in vitro neural cell systems.</li>
	</ol>
	</li>
	<li>Harvesting cells7:
	<ol>
		<li>Gently wash the dish/flask of adherent cells with Mg2+/ Ca2+ free phosphate-buffered saline (PBS) at RT (e.g., 5 ml for T75 flask, 3 ml per well for 6-well plate, or 10 ml for 10 cm dish). 
		NOTE: The PBS used throughout the protocol is Mg2+/ Ca2+ free.
		<ol>
			<li>For the video example, use a T75 flask of SH-SY5Y neuroblastoma cells at 80% confluency. Apply additional washing steps in cases where considerable debris is present in the dish.</li>
			<li>Consider washes with serum albumin-containing PBS48, Percoll, or Ficoll49 centrifugation gradients and/or commercially available beads50,51. Removal of myelin and other lipid or other contaminants is critical, particularly when adult primary tissue sources are being used.</li>
		</ol>
		</li>
		<li>Add pre-warmed (37 °C) trypsin replacement at an appropriate volume that covers the entire surface of the tissue culture vessel. 
		NOTE: Alternatively, consider Accutase or other enzymatic digestion options. This critical step may negatively affect surface epitope expression (see 7).</li>
		<li>Incubate the dish/flask at 37 °C for 2 - 5 min (depending on the cell type) to allow cells to detach. Gently tap the tissue culture vessel or flush with a serological pipet to dislodge the cells. Avoid over digestion (as this can cause cell loss and clotting at later steps).</li>
		<li>Quench the trypsin replacement by adding twice the volume of flow buffer (2% FBS in PBS) and collect the cells in a 15 ml conical tube.</li>
		<li>Gently triturate the cell suspension using a microliter pipet (100 - 1,000 µl) or a 5 ml serological pipet to prepare a single cell suspension.</li>
		<li>Centrifuge the cells at 220 x g for 5 min at 25 °C. Carefully aspirate the supernatant leaving the pellet behind.</li>
		<li>Re-suspend the pellet in an appropriate volume of flow buffer, depending on the size of the pellet (e.g., for one confluent T75 flask of SH-SY5Y cells the typical yield is at least 10 x 106 cells, in which case the cells are resuspended in 5 ml of flow buffer). 
		NOTE: If larger chunks or clotting are observed, filter through a 30 - 100 µm mesh.</li>
	</ol>
	</li>
	<li>Cell counting52:
	<ol>
		<li>Transfer a small aliquot of the cell suspension to a microcentrifuge tube and dilute at a defined ratio in a volume of trypan blue or an alternative viability dye before transfer to a hemocytometer or automated cell counting system.</li>
		<li>Dilute the cell suspension to a concentration of 1 x 106 viable cells/ml by adding the appropriate volume of flow buffer or PBS with 0.1% BSA (if proceeding with CFSE labeling). 
		NOTE: Propidium iodide, 7-aminoactinomycin D, annexin V and commercially available fixable viability assay kits represent alternative options in order to assess the viability of cells. Also, apoptosis assays using caspase-3 fluorescence as described previously53 may be used. Fluorescent channels will be “occupied” by these reagents which may limit the options for subsequent steps if included in all samples.</li>
	</ol>
	</li>
</ol>
2. Intracellular Dye Labeling Using CFSE (Figure 3)
<ol>
	<li>Dilute the CFSE to a desired stock concentration that can be easily used. 
	NOTE: For these experiments a stock concentration of 0.01 mM was used. Determine the optimal working concentration of CFSE empirically.</li>
	<li>Add 10 µl of 0.01 mM CFSE solution per ml of cells (Section 1.3.2, concentration of 1 x 106 cells/ml in PBS + 0.1% BSA) for a final concentration of 0.1 µM. Briefly vortex to mix well. 
	NOTE: CFSE concentrations used here are about ten-fold lower than the ones commonly applied in proliferation assays, hence cell toxicity of the dye is minimal. We do not observe negative effects on cell viability.</li>
	<li>Incubate for 5 min at RT with constant shaking (200 rpm). Protect from light.</li>
	<li>Quench the dye by adding 5 volumes of flow buffer to the tubes. Centrifuge at 94 x g for 5 min at RT.</li>
	<li>Discard the supernatant leaving the pellet behind. Re-suspend the cells with 5 volumes of flow buffer.</li>
	<li>Centrifuge at 94 x g for 5 min at RT. Discard the supernatant and re-suspend the cells in flow buffer at a concentration of 1 x 106 cells/ml.</li>
	<li>Add an equal number of unstained cells of interest to the stained cell suspension. Proceed to surface antigen staining protocol (Section 3).</li>
</ol>
<img alt="Flow cytometry scatter plots, CFSE vs CD24/CD54, cell population analysis, SH-SY5Y and BJ cells." src="/files/ftp_upload/52241/52241fig3highres.jpg" width="700" data-altupdatedat="1747911167000" data-alt="Flow cytometry analysis; CFSE vs CD24/CD54 expression in SH-SY5Y and BJ cells; scatter plots."> 
Figure 3. Detection of differential CD surface antigen expression between two cell lines via CFSE dye labeling. SH-SY5Y neuroblastoma cells are pre-labeled with CFSE for subsequent identification in comparison to unlabeled BJ fibroblasts. Co-staining of the mixed sample (right panels) with surface markers CD24 or CD54 (both conjugated to APC) demonstrates that cell lines are easily distinguishable due to the CFSE staining (arrows = SH-SY5Y; arrowheads = BJ fibroblasts). The majority of SH-SY5Y cells express CD24 but not CD54 (ICAM-1). In contrast, BJ fibroblasts (CFSE-negative) are positive for CD54 but largely negative for CD24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
3. Cell Surface Staining
<ol>
	<li>Label tubes including samples and critical controls (see Table 1).</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tube no.</td>
			<td>Sample name</td>
			<td>Antigen-fluorophore</td>
			<td>Dilution</td>
		</tr>
		<tr>
			<td>1</td>
			<td>Unstained cells</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>2</td>
			<td>Single stained</td>
			<td>CD24-APC</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td>3</td>
			<td>Single stained</td>
			<td>TUJ1-Alexa fluor 488nm</td>
			<td>1:2,000</td>
		</tr>
		<tr>
			<td>4</td>
			<td>Double stained</td>
			<td>CD24-APC</td>
			<td>1:50</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td>TUJ1-Alexa fluor 488nm</td>
			<td>1:2,000</td>
		</tr>
		<tr>
			<td>5</td>
			<td>Single stained</td>
			<td>Secondary only: Alexa fluor 488nm</td>
			<td>1:2,000</td>
		</tr>
	</tbody>
</table>
Table 1. List of tubes to be included in a typical flow cytometry experiment. The table shows a minimal set of sample tubes required for a co-staining experiment described in this video article. An ideal experiment needs to include all necessary controls (negative, positive as well as compensation controls) for accurate interpretation of the results obtained.
<ol start="2">
	<li>Add 100 µl of cell suspension (from Section 2.7 or 1.3.2) to each 1.5 ml microcentrifuge tube. 
	NOTE: Make sure a minimum of 0.1 x 106 cells are present per 100 µl of cell suspension.</li>
	<li>Add fluorophore conjugated antibody to the sample at an appropriate dilution. 
	NOTE: Determine working dilution for each antibody prior to the experiment. See Table 2 for a list of neural surface antigens.</li>
</ol>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Antigen</td>
			<td>Cell Type</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>CD15</td>
			<td>Neural stem cells </td>
			<td>[28, 67]</td>
		</tr>
		<tr>
			<td>CD24</td>
			<td>Neuronal cells</td>
			<td>[28, 68]</td>
		</tr>
		<tr>
			<td>CD29</td>
			<td>Neural stem cells</td>
			<td>[28, 69, 70]</td>
		</tr>
		<tr>
			<td>CD44</td>
			<td>Glial cells</td>
			<td>[25]</td>
		</tr>
		<tr>
			<td>CD49f</td>
			<td>Neural stem cells </td>
			<td>[38]</td>
		</tr>
		<tr>
			<td>CD56 (NCAM)</td>
			<td>Neuronal cells</td>
			<td>[71]</td>
		</tr>
		<tr>
			<td>CD133</td>
			<td>Neural stem cells </td>
			<td>[27]</td>
		</tr>
		<tr>
			<td>CD184</td>
			<td>Neural stem cells and glial cells</td>
			<td>[25]</td>
		</tr>
		<tr>
			<td>CD200</td>
			<td>Neuronal cells</td>
			<td>[38]</td>
		</tr>
		<tr>
			<td>CD271</td>
			<td>Neural crest stem cells</td>
			<td>[25]</td>
		</tr>
		<tr>
			<td>A2B5</td>
			<td>Glial cells</td>
			<td>[31]</td>
		</tr>
		<tr>
			<td>CORIN</td>
			<td>Dopaminergic precursors</td>
			<td>[35, 36]</td>
		</tr>
		<tr>
			<td>FORSE1</td>
			<td>Neural stem cells (NSC)</td>
			<td>[72 ]</td>
		</tr>
		<tr>
			<td>GLAST</td>
			<td>Glial cells</td>
			<td>[33]</td>
		</tr>
		<tr>
			<td>NG2</td>
			<td>Glial cells</td>
			<td>[32]</td>
		</tr>
	</tbody>
</table>
Table 2. Selection of neural surface antigens. This table provides a list of surface epitopes found to be expressed by various neural cell types to exemplify the increasing panel of surface antigens used to characterize the neural lineage. Note that this selection is far from being complete and that most of these markers are also expressed by a range of other neural and non-neural cells. Consequently, combinations of several markers will be required to better define and isolate the indicated neural subsets.
<ol start="4">
	<li>Incubate for 30 min on an orbital shaker (200 rpm) in the dark.</li>
	<li>Flow buffer wash
	<ol>
		<li>Add 1 ml of flow buffer to the tubes. Centrifuge at 380 x g for 4 min at 4 °C.</li>
		<li>Discard the supernatant leaving the pellet behind.</li>
	</ol>
	</li>
	<li>Repeat the wash step again.</li>
	<li>After the second wash, decant the supernatant and re-suspend the cells in flow buffer to a final volume of 100 µl.</li>
	<li>Use sample for flow cytometric analysis. Alternatively, proceed with Section 4 and 5. 
	NOTE: If cells are to be sorted and put back into culture post-FACS (i.e., viable cell suspension without fixation or permeabilization), apply aseptic techniques during the harvest, staining and analytical steps.</li>
</ol>
4. Fixation and Permeabilization38
<ol>
	<li>Fixation using paraformaldehyde (PFA):
	<ol>
		<li>Prepare fixation buffer containing 2% PFA in PBS. 
		NOTE: PFA is harmful to humans and the environment. Use appropriate personal protective equipment and discard waste in accordance with local regulations.</li>
		<li>Add 500 µl of fixation buffer to 100 µl of cell suspension.</li>
		<li>Incubate the tubes at RT for 15 min on an orbital shaker (100 rpm) in the dark.</li>
	</ol>
	</li>
	<li>PBS wash:
	<ol>
		<li>Add 1 ml of PBS to the tube. Centrifuge at 380 x g for 3 min at 4 °C.</li>
		<li>Discard/ decant the supernatant leaving approximately 100 µl in the tube.</li>
	</ol>
	</li>
	<li>Permeabilization with Tween-20:
	<ol>
		<li>Prepare permeabilization buffer containing 0.7% Tween-20 in PBS.</li>
		<li>Add 500 µl of permeabilization buffer to 100 µl of cell suspension.</li>
		<li>Incubate the tubes at RT for 15 min on an orbital shaker (100 rpm) in the dark.</li>
	</ol>
	</li>
	<li>Wash the cells once with PBS (as described in Section 4.2) and remove the supernatant from the tubes completely, leaving only the pellet behind.</li>
</ol>
5. Intracellular Antigen Staining38 (Figure 4)
<ol>
	<li>Preparation of primary antibody solutions:
	<ol>
		<li>Dilute the primary antibodies in dilution buffer containing 1% bovine serum albumin, 10% serum (e.g., normal donkey or goat serum) and 0.5% Tween-20 in PBS. 
		NOTE: Choose the serum to be used depending on the species the secondary antibodies were raised in.</li>
		<li>Alternatively, use Zenon fluorescein labeling of the primary antibody according to the manufacturer’s instructions.
		<ol>
			<li>Prepare 1 µg of primary antibody in PBS at an appropriate dilution (total volume ≤ 20 µl).</li>
			<li>Add 5 µl of Zenon fluorescein IgG labeling reagent (Component A) to the antibody solution.</li>
			<li>Incubate the mixture for 5 min at RT.</li>
			<li>Add 5 µl of Zenon blocking reagent (Component B) to the reaction mixture.</li>
			<li>Incubate the mixture for 5 min at RT. Apply the antibody to the sample within 30 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Primary antibody staining:
	<ol>
		<li>Add 100 µl of the primary antibody solution to the cell pellet and gently triturate to mix.</li>
		<li>Alternatively, add Zenon fluorescein labeled antibody to the cell suspension at the appropriate dilution.</li>
		<li>Incubate the tubes at RT for 30 min on an orbital shaker (200 rpm), protected from light. Wash the cells once with PBS (as describes in Section 4.2) and remove the supernatant from the tubes completely, leaving only the pellet behind.</li>
	</ol>
	</li>
	<li>Secondary antibody staining (not required for the Zenon fluorescein labeled antibodies):
	<ol>
		<li>Dilute the secondary antibodies in PBS at an appropriate concentration.</li>
		<li>Add 100 µl of the secondary antibody solution to the cell pellet and gently triturate to mix. Incubate the tubes at RT for 30 min on a shaker (200 rpm) in the dark.</li>
	</ol>
	</li>
	<li>Wash the samples two times with PBS (as described in Section 4.2).</li>
	<li>Wash once with flow buffer (see Section 3.5).</li>
	<li>Re-suspend the cells in approximately 150 µl of flow buffer and analyze on the flow cytometer.</li>
</ol>
<img alt="Flow cytometry dot plots showing MAP2 and CD24 staining, fluorescence intensity comparison." src="/files/ftp_upload/52241/52241fig4highres.jpg" width="700" data-altupdatedat="1747911167000" data-alt="Flow cytometry scatter plots showing FITC vs APC, analyzing MAP2-AF488 and CD24-APC staining."> 
Figure 4. Co-staining of surface and intracellular proteins. Flow cytometry data exemplifies a comparison between primary + secondary antibody-based versus Zenon fluorescein-based intracellular staining in combination with surface staining. Exclusive positivity on the y-axis (upper left quadrant) and the x-axis (lower right quadrant) shows cells stained for MAP2 and CD24, respectively. After co-staining, shared MAP2 and CD24 expression can be seen in the upper right quadrant (right panels). Comparison of using Alexa fluor 488 (top; AF488) versus Zenon fluorescein (bottom) for MAP2-labeling yields similar results. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
6. Flow Cytometric Analysis
<ol>
	<li>Conduct flow cytometric analysis immediately after the completion of the staining protocol using a flow cytometer with appropriate filters for signal detection. Use 488 nm blue and 640 nm red laser with FL-1 (533/30), FL-2 (585/40), and FL-4 (675/25) bandpass filters.</li>
	<li>Set up primary gates based on the forward and side scatter excluding debris and dead cells.</li>
	<li>Set fluorescence gates for surface and intracellular antigen to ≤0.5% based on the unstained samples and compensation for spectral overlap using single stained controls.</li>
</ol>

The authors declare no potential conflicts of interest.

The protocol presented here is well established for neural cell cultures derived from human stem cells, but can be equally applied to other neural cell sources including primary tissue or neural cell lines. In addition to embryonic sources, neural stem or progenitor cells can be extracted from the neurogenic regions of adult brain27. Moreover, flow cytometry and FACS can be exploited to quantify, analyze and isolate various cell populations including mature neurons54, astroglia33, microglial55, oligodendrocytes56, or endothelial cells57 from postnatal brain tissue.
Regardless of the source, optimal conditions for fixation and permeabilization need to be determined for each cell type experimentally, as excess of either of the steps could lead to a massive change of scatter properties affecting flow cytometric readout. Efficiency of the protocol also depends on the stability of the surface molecules post permeabilization. It should be noted that different surface epitopes can be affected by cell manipulation as part of the procedure to varying degrees. Also enzymatic harvesting with trypsin replacement or liberase-1, for instance, may affect a range of cell surface epitopes but to a lesser degree compared to the usage of Accutase or papain7.
Specific epitopes may be affected by the same treatment paradigm to a varying degree: while VCAM-1 (Vascular cell adhesion protein-1; CD106) can be affected by harvesting, dissociation, fixation and permeabilization in a quite sensitive manner, ICAM-1 (Intercellular Adhesion Molecule-1; CD54) may display a more robust expression pattern preserved throughout the procedure58.
While previously either surface25,28 or intracellular antigen labeling14 have been applied in neurobiology paradigms, approaches combining both methods38 can yield novel neural surface antigen candidates and provide additional cytometric readout options.
We routinely conduct the surface staining step prior to intracellular antigen staining and have used that strategy to expand our set of surface markers for neuropoiesis38 using a range of human neural cell lines. Titration of antibodies and incubation times will need to be optimized for the respective cell types, target epitopes and experimental paradigm. Similarly, the CFSE concentration and the amount of cells used per staining must be optimized for each cell line, as not all cell lines stain in an identical fashion with the same amount of CFSE concentration.
CFSE has a peak excitation of 494 nm and peak emission of 521 nm (green channel; FL-1 detector), hence any fluorescence spillover to other channels needs to be compensated to determine specific fluorescence emission. Alternative dyes such as Cell Trace Violet (CTV), Cell Proliferation Dye eFluor 670 (CPD), Horizon V550, Horizon V450 have been successfully tried and tested to be used instead of CFSE59,60.
In flow cytometry, it is critical to determine the actual specific fluorescent signal from background. Therefore, the most appropriate controls have to be carefully chosen, and may include using a non-neural or otherwise negative cell line to underline antibody specificity (so called internal negative controls38,46). Autofluorescence of each cell source must be taken into account. Isotype controls can be considered largely dispensable in most neural cell detection paradigms13,46.
Given the considerable variation observed in cell culture systems (particularly those derived from stem cells) and of the staining procedure, the inclusion of biological replicates is strongly recommended. It should be noted that usage of genetic reporters is another way to use neural flow cytometry61,62. Recently, Maroof et al. used an Nkx2.1-GFP reporter approach to sort distinct cortical-like neuronal subpopulations from human embryonic stem cells63.
An up-and-coming field is represented by the exploration of neural exosomes in biomedical areas as diverse as tumor biology and neurodegeneration64, and flow cytometry of exosomes or their isolation by bead-based assays provides important tools for this quest65. For related video protocol see66.

Flow cytometry has been extensively exploited in immunology, hematology and oncology to define cell populations via intrinsic scatter properties, cell surface antigen expression, and other fluorescence parameters1-3. Our insights into blood lineage development and disease are a result to a significant degree of the continuous refinement of this methodology after its initial implementation4,5. Increased awareness of the quantitative and overall analytic potential of flow cytometry has recently encouraged its more widespread use in stem cell research and may enable similarly profound progress in a shorter time frame6. However, the application of flow cytometry to specifically analyze and isolate neural populations has long been perceived as challenging. In contrast to hematopoietic cells that naturally exist in suspension, neural cell types are typically harvested from excessively complex sources that may include glia and various other surrounding cells as well as an intricate network of process-bearing neurons. Consequently, neurobiology has yet to implement the versatility of flow cytometry to its complete potential in daily research routines. However, as long as a viable single cell suspension can be generated (and protocols have been devised and optimized for that purpose7), flow cytometry and fluorescence-activated cell sorting (FACS) can be considered a valuable element of the analytical repertoire in neurobiology8-11.
<img alt="Flow cytometry diagram, single cell analysis, fluidics, optics, scatter detection, fluorescence." src="/files/ftp_upload/52241/52241fig1highres.jpg" width="600" data-altupdatedat="1747911167000" data-alt="Flow cytometry diagram, showing cell suspension in flow cytometer for fluorescence detection."> 
Figure 1. Principle of flow cytometric analysis and components of a flow cytometer. Flow cytometers comprise three main systems: fluidics, optics and electronics. A streamlined flow of cells in suspension (prepared from primary tissue or in vitro culture) is accomplished by the sheath fluid via hydrodynamic focusing, restricting the sample to its center core. The optical parts are composed of lasers that illuminate the stream of cells and optical filters that direct the signal to the appropriate detectors. The light signals detected are converted to electronic signals, subsequently processed by a computer and visualized on a monitor for data analysis and gating. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Users of flow cytometric methods profit from at least a basic understanding of the underlying fundamentals including a cytometer’s building blocks (for review see12,13; also see Figure 1). A laser beam intersects with a hydrodynamically focused fluidic stream that contains the cells in suspension, which in turn pass through the laser beam in ‘single file’ one after another. The interception of a cell (or any other particle, for that matter) with the laser results in the scattering of light from this interrogation point. Scattered light can be detected in continuation of the laser direction (forward scatter, associated with the size of the particle), as well as perpendicular to its direction (side scatter; reflecting the granulosity of the particle/cell). These aforementioned scatter properties do not require specific labeling, which is why an unlabeled sample (or also cellular debris, air bubbles, etc.) will generate a signal (event) on the bivariate forward scatter versus side scatter plot commonly used for initial gating. By using the appropriate lasers and filters specific for the corresponding excitation and emission spectra, a cell can be analyzed for its positivity, level of intensity, or absence of fluorescent markers. The majority of flow cytometric applications have focused on characterization via cell surface antigens. Unlike the hematopoietic lineage, the neural lineage has remained less extensively defined according to surface epitope expression patterns5. One advantage of exploiting surface antigens is that live cells can be subjected to cell sorting paradigms such as FACS. In contrast, intracellular antigen staining requires fixation and permeabilization steps to mediate the epitope-antibody interaction, precluding downstream applications that require viable cells. Of note, such approaches still allow for numerous quantitative assays14 as well as downstream analyses for RNA and protein expression15. Hematology, immunology and oncology have often used more than a dozen markers in conjunction to define particular subpopulations16. Additionally, mass cytometry or CyTOF can now be used to analyze up to 30 parameters simultaneously17,18.
For neural stem cell applications as well as primary cultures14,19,20 the heterogeneity of cells in vitro is a common phenomenon21-23. The cells not representing the target population of interest embody a potentially confounding factor for experimental readout24,25. Conveniently, the different cellular subsets present within a heterogeneous cell suspension bear distinct (known or yet to be deciphered) antigen expression profiles, which can be utilized to define these various populations. Flow cytometry can hence play a crucial role in resolving cellular heterogeneity and, thereby, facilitate biomedical applications (in vitro assays, cell therapy) and optimize quantitative readout by focusing on the most relevant subset24,26. Various surface antigen combinations have been identified over the past few years to allow the quantitation and isolation of specific neural cell types. This includes CD133 for the enrichment of neural stem cells27, a combination of the CD15/CD24/CD29 surface antigens for the isolation of NSC, differentiated neuron and neural crest cells28 or CD15/CD24/CD44/CD184/CD271 to isolate neural and glial subsets25, among other signatures29,30. Beyond neurons, glial markers include A2B531, CD4425, NG232 and GLAST33. A recent publication has exploited the midbrain floorplate precursor marker CORIN34,35 to enrich for dopaminergic precursors in Parkinson cell transplantation paradigms36. CD molecules are not only markers, but functionally relevant mediators of cell-cell interactions and of a cell’s ability to respond to cues from extracellular matrix molecules and growth factors37. One strategy of further enhancing the arsenal of combinatorial CD antigens to characterize neural lineage development is to use known intracellular markers to screen for and define CD antigen combinations for a particular cell type of interest. We have recently exploited such an approach and identified CD49f–/CD200high combinatorial expression patterns as a novel approach for enriching neuronal subsets from neurally differentiated induced pluripotent stem cell culture systems38. Here, we include and discuss the latter protocol (and optional variations thereof) in which surface staining and intracellular staining can be used simultaneously for defining neural cell subpopulations by flow cytometry.
<img alt="Flowchart for FACS process: neural cell culture to cell sorting, CFSE dye, antigen stain, analysis." src="/files/ftp_upload/52241/52241fig2highres.jpg" width="300" data-altupdatedat="1747911167000" data-alt="Neural cell flow chart: culture to FACS, CFSE dye, antigen stain, cytometric analysis process."> 
Figure 2. Flow diagram of experimental protocol options. The figure depicts a schematic representation of the major steps involved in the protocol. Optional steps (CFSE dye or intracellular antigen labeling) are indicated by light grey boxes. After harvesting, it is essential to assess the viability and cell number of neural cell suspensions prior to cell surface staining. Positive as well as negative controls need to be included in addition to the samples of interest. Samples can be analyzed by flow cytometric analysis and/or used in cell sorting paradigms. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52241/52241fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
While we have previously used primary antibody in combination with secondary antibody for intracellular staining38, we now introduce non-covalent labeling of the primary antibody via fluorescent Fab fragments (Zenon labeling) as a slight variation, thereby reducing the steps of cell manipulation39. Moreover, as a further example of the protocol’s versatility, we employ an optional labeling of one experimental subset by carboxyfluorescein succinimidyl ester (CFSE) prior to surface antigen staining. Such CFSE pre-labeling enables the immediate direct comparison of two cell lines or experimental conditions (CFSE-labeled vs. unlabeled) within a single sample tube, reducing variance or subtle differences in incubation time and saving antibody. CFSE is an established fluorescent dye that is commonly used for cell tracking40, in proliferation41,42 and barcoding experiments43,44. Finally, while actual sorting steps (FACS, immunomagnetic cell separation or immunopanning) are not part of this protocol, in principle, the harvesting and labeling procedures described here do yield samples that can be subjected to surface antigen- or intracellular labeling-based sorting applications15 25,28.
With this article, we aim to: summarize a viable surface antigen staining protocol25,28, summarize a protocol for detection of intracellular targets as well as combined surface and intracellular antigen analysis38, present an intracellular CFSE dye labeling step41,45 as an experimental option for comparative analyses of neural cell populations, and summarize approaches to flow cytometric analysis (appropriate controls13,46, gating strategy and data presentation47).

<table><tbody><tr><td>DMEM/F12 (1:1) (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12)</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>11330057</td><td></td></tr><tr><td>DPBS without Ca&lt;sup&gt;2+&lt;/sup&gt; Mg&lt;sup&gt;2+&lt;/sup&gt;</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>14190169</td><td></td></tr><tr><td>Fetal bovine serum, qualified, E.U.-approved, South America origin (FBS)</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>10270-106</td><td></td></tr><tr><td>MEM Non-essential amino acids (100x)</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>11140035</td><td></td></tr><tr><td>TrypLE Express</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>12604013</td><td></td></tr><tr><td>Trypan blue solution, 0.4%</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>15250061</td><td></td></tr><tr><td>Paraformaldehyde</td><td><a href="http://www.carlroth.com" target="_blank"><img src="/img/new_window.png"> Carl Roth</a></td><td>335.3</td><td></td></tr><tr><td>Bovine serum albumin (BSA) Fraction V</td><td><a href="http://www.gelifesciences.com" target="_blank"><img src="/img/new_window.png"> PAA Laboratories, Coelbe</a></td><td>K41-001</td><td></td></tr><tr><td>Tween-20 Detergent</td><td><a href="http://www.merckmillipore.de" target="_blank"><img src="/img/new_window.png"> Calbiochem</a></td><td>655205</td><td></td></tr><tr><td>Carboxyfluorescein succinimidyl ester (CFSE)</td><td><a href="http://www.ebioscience.com" target="_blank"><img src="/img/new_window.png"> eBioscience</a></td><td>65-0850-84</td><td></td></tr><tr><td>DMSO</td><td><a href="http://www.applichem.com" target="_blank"><img src="/img/new_window.png"> AppliChem</a></td><td>A1584</td><td></td></tr><tr><td>Bottle top filters express plus 0.22 &amp;micro;m, 250 ml</td><td><a href="http://www.millipore.com" target="_blank"><img src="/img/new_window.png"> Millipore</a></td><td>SCGPU02RE</td><td></td></tr><tr><td>Cell culture treated flasks (T 25)</td><td><a href="http://www.thermoscientific.com" target="_blank"><img src="/img/new_window.png"> NUNC</a></td><td>156367</td><td></td></tr><tr><td>Cell culture treated flasks (T 75)</td><td><a href="http://www.thermoscientific.com" target="_blank"><img src="/img/new_window.png"> NUNC</a></td><td>156499</td><td></td></tr><tr><td>Conical tubes (15 ml)</td><td><a href="http://www.greinerbioone.com" target="_blank"><img src="/img/new_window.png"> Greiner Bio-One</a></td><td>188271</td><td></td></tr><tr><td>Conical tubes (50 ml)</td><td><a href="http://www.greinerbioone.com" target="_blank"><img src="/img/new_window.png"> Greiner Bio-One</a></td><td>227261</td><td></td></tr><tr><td>Pasteur pipet, glass (150 mm)</td><td><a href="http://www.stein-labortechnik.de" target="_blank"><img src="/img/new_window.png"> STEIN Labortechnik, Remchingen</a></td><td>S03710150</td><td></td></tr><tr><td>Pipet tips (0.1-10 &amp;micro;l)</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>4125</td><td></td></tr><tr><td>Pipet tips (1-200 &amp;micro;l)</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>4126</td><td></td></tr><tr><td>Pipet tips (100-1000 &amp;micro;l)</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>4129</td><td></td></tr><tr><td>Serological pipets, 5 ml</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>4051</td><td></td></tr><tr><td>Serological pipets, 10 ml</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>4101</td><td></td></tr><tr><td>Serological pipets, 25 ml</td><td><a href="http://www.corning.com" target="_blank"><img src="/img/new_window.png"> Corning</a></td><td>4251</td><td></td></tr><tr><td>Microcentrifuge tubes (0.5 ml)</td><td><a href="http://www.sarstedt.com" target="_blank"><img src="/img/new_window.png"> Sarstedt</a></td><td>72,699</td><td></td></tr><tr><td>Microcentrifuge tubes (1.5 ml)</td><td><a href="http://www.greinerbioone.com" target="_blank"><img src="/img/new_window.png"> Greiner Bio-One</a></td><td>616201</td><td></td></tr><tr><td>Microcentrifuge tubes (2.0 ml)</td><td><a href="http://www.sarstedt.com" target="_blank"><img src="/img/new_window.png"> Sarstedt</a></td><td>72,695,500</td><td></td></tr><tr><td>Anti-Human CD24 APC monoclonal antibody</td><td><a href="http://www.ebioscience.com" target="_blank"><img src="/img/new_window.png"> eBioscience</a></td><td>17-0247-42</td><td>Working dilution 1:50</td></tr><tr><td>Anti-Human CD54 PE monoclonal antibody</td><td><a href="http://www.ebioscience.com" target="_blank"><img src="/img/new_window.png"> eBioscience</a></td><td>12-0549-42</td><td>Working dilution 1:50</td></tr><tr><td>Neuronal Class III &amp;beta;-Tubulin (Tuj1) polyclonal antibody</td><td><a href="http://www.covance.com" target="_blank"><img src="/img/new_window.png"> Covance</a></td><td>PRB-435P</td><td>Working dilution 1:2,000</td></tr><tr><td>Alexa Fluor 488 Donkey anti Rabbit</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>A21206</td><td>Working dilution 1:2,000</td></tr><tr><td>Zenon&amp;reg; Fluorescein Rabbit IgG Labeling Kit</td><td><a href="http://www.lifetechnologies.com" target="_blank"><img src="/img/new_window.png"> Life Technologies</a></td><td>Z-25342</td><td></td></tr><tr><td>Neubauer-Improved counting chamber</td><td><a href="http://www.marienfeld-superior.com" target="_blank"><img src="/img/new_window.png"> Marienfeld</a></td><td>0640010</td><td></td></tr><tr><td>Vortex</td><td><a href="http://www.scientificindustries.com" target="_blank"><img src="/img/new_window.png"> Scientific Industries</a></td><td>G560E</td><td></td></tr><tr><td>Thermomixer comfort</td><td><a href="http://www.eppendorf.com" target="_blank"><img src="/img/new_window.png"> Eppendorf</a></td><td>5355 000.001</td><td></td></tr><tr><td>Accuri C6 flow cytometer</td><td><a href="http://www.bdbiosciences.com/instruments/accuri" target="_blank"><img src="/img/new_window.png"> Becton Dickinson (BD)</a></td><td>653118</td><td></td></tr><tr><td>Microcentrifuge refrigerated, PerfectSpin 24 R</td><td><a href="http://www.peqlab.de" target="_blank"><img src="/img/new_window.png"> Peqlab</a></td><td>91-PSPIN-24R</td><td></td></tr><tr><td>Orbital shaker, Unimax 1010</td><td><a href="http://www.heidolph-instruments.de" target="_blank"><img src="/img/new_window.png"> Heidolph</a></td><td>543-12310-00</td><td></td></tr><tr><td>Centrifuge refrigerated, Rotanta 96 RC</td><td><a href="http://www.hettichlab.com" target="_blank"><img src="/img/new_window.png"> Hettich</a></td><td>4480-50</td><td></td></tr><tr><td>Class II Biological safety cabinet Safe 2020</td><td><a href="http://www.thermoscientific.com" target="_blank"><img src="/img/new_window.png"> Thermo Scientific</a></td><td>51026640</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Incubator, Heracell 240i</td><td><a href="http://www.thermoscientific.com" target="_blank"><img src="/img/new_window.png"> Thermo Scientific</a></td><td>51026331</td><td></td></tr><tr><td>Vacuum system, Vacusafe comfort</td><td><a href="http://www.integra-biosciences.de" target="_blank"><img src="/img/new_window.png"> Integra Biosciences</a></td><td>158320</td><td></td></tr><tr><td>Microscope, Axiovert 40 CFL</td><td><a href="http://www.zeiss.de" target="_blank"><img src="/img/new_window.png"> Zeiss</a></td><td>451212</td><td></td></tr><tr><td>Pipet controller, accu-jet pro</td><td><a href="http://www.brand.de" target="_blank"><img src="/img/new_window.png"> Brand</a></td><td>26303</td><td></td></tr><tr><td>Micropipet, Pipetman neo P20N (2-20 &amp;micro;l)</td><td><a href="http://www.gilson.com" target="_blank"><img src="/img/new_window.png"> Gilson</a></td><td>F144563</td><td></td></tr><tr><td>Micropipet, Pipetman neo P200N (20-200 &amp;micro;l)</td><td><a href="http://www.gilson.com" target="_blank"><img src="/img/new_window.png"> Gilson</a></td><td>F144565</td><td></td></tr><tr><td>Micropipet, Pipetman neo P1000N (100-1000 &amp;micro;l)</td><td><a href="http://www.gilson.com" target="_blank"><img src="/img/new_window.png"> Gilson</a></td><td>F144566</td><td></td></tr></tbody></table>

flow cytometry protocols for surface and intracellular antigen analyses of neural cell types

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of protocols for flow cytometric analysis of the cluster of differentiation (CD) surface antigens and intracellular antigens in neural cell types. Our step-by-step description of the methodological procedures include: the harvesting of neural in vitro cultures, an optional carboxyfluorescein succinimidyl ester (CFSE)-labeling step, followed by surface antigen staining with conjugated CD antibodies (e.g., CD24, CD54), and subsequent intracellar antigen detection via primary/secondary antibodies or fluorescently labeled Fab fragments (Zenon labeling). The video demonstrates the most critical steps. Moreover, principles of experimental planning, the inclusion of critical controls, and fundamentals of flow cytometric analysis (identification of target population and exclusion of debris; gating strategy; compensation for spectral overlap) are briefly explained in order to enable neurobiologists with limited prior knowledge or specific training in flow cytometry to assess its utility and to better exploit this powerful methodology.

The protocol presented here allows for versatile experimental approaches (Figure 2). In its shortest version (steps 1, 3 and 6), it can be considered a guide for simple staining of surface antigens.&#160; In its more complex form, a number of co-labeling paradigms with a range of intracellular antigens can be pursued (optional steps 2 and/or 4 to 5). Moreover, the CFSE-labeling step exemplifies its utility for cell labeling/tracking of subpopulations in cell-cell interaction studies or flow cytometric barcoding experiments (step 2). Viable cell populations (obtained from steps 2 and/or 3) are amenable to cell sorting paradigms (FACS) and downstream analysis including post-sort cell culture. On the other hand, the combined intracellular and surface antigen staining (steps 3 to 5) provides analytical readout opportunities in its own right. For example, identifying co-localization of certain CD antigens on particular subsets of neural cell populations (applied by us in Tura&#231; et al.38 to identify CD49f&#8211;/CD200high signatures for neuronal FACS). These and other example neural markers are provided in Table 2, of which CD15, CD29, CD49f and FORSE1, are primarily associated with neural stem cells, and CD24 and CD56 are abundant on neuronal cells. While few of these markers are exclusive, different staining intensities and multivariate expression analysis can help to identify specific marker sets for analysis and isolation of particular neural subpopulations.
Figure 3 exemplifies typical results obtained&#160;from the combinatorial usage of intracellular dye and surface staining. The data show how two distinct cell populations can easily be distinguished in a mixed sample due to the prior labeling of one of the populations. Here, SH-SY5Y neuroblastoma cells were labeled with CFSE, and exhibit fluorescence in the green channel (FL1; y-axis) that is clearly separate from the unstained second population, in this case BJ fibroblasts. While well-established characteristic fibroblast (CD54) and neural (CD24) markers are shown (present on their respective target populations; FL2 or FL4 fluorescence, respectively; x-axis) analogous approaches can be exploited to screen for additional unidentified antigens. Furthermore, the effects of cell-cell interactions of two (or more) subpopulations that were labeled prior to co-culture experiments can be studied in this manner.

Figure 4 shows the unstained SH-SY5Y cells as well as samples stained for CD24-APC and/or the intracellular neuronal MAP2 antigen (FITC channel) after fixation and permeabilization. As the latter can affect scatter properties, background fluorescence and surface epitope staining, the plots serve to document maintained surface expression of neural CD24 after intracellular staining. Moreover, the co-localization of neuronal MAP2 staining on the CD24-positive fraction is shown. It is critical to ensure faithful detection of the surface antigen by comparison to CD24 surface expression on live cells. Moreover specificity of the intracellular detection over background, non-specific antibody binding and autofluorescence needs to be confirmed. Detection of intracellular antigens via Zenon fluorescence-based or primary + secondary antibody-based strategies yields comparable results, which provides a rationale for considering this more time-efficient approach (skipping step 5.3). While the protocol is robust and enables the combined detection of a range of surface and intracellular markers with limited background fluorescence, it is recommended to closely adhere to the incubation times and washing steps provided in the protocol. Moreover, it is advisable to analyze at least three independent experimental repeats and to note that labeling intensity and surface epitope stability can be marker-specific and may require further optimization. Once established for a particular target, despite lacking the ability to analyze morphological features, flow cytometric detection of surface and intracellular antigens or intracellular antigens alone is compatible with conventional fluorescence microscopy.

Watch this Scientific Journal Video about Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types at JoVE.com

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Flow cytometry has been extensively used to define cell populations in immunology, hematology and oncology. Here, we provide a detailed description of ...

flow-cytometry-protocols-for-surface-intracellular-antigen-analyses

Nanyang Technological University

Research

JoVE Journal

Neuroscience

52.2K Views.  University of Freiburg. We provide a detailed description of a protocol for flow cytometric analysis of surface antigens and/or intracellular antigens in neural cell types. Critical aspects of experimental planning, step-by-step methodological procedures, and fundamental principles of flow cytometry are explained in order to enable neurobiologists to exploit this powerful technology.

Video: Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

Medicine

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...

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

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

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.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Immunology and Infection

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Flow Cytometric Analysis of Multiple Mitochondrial Parameters in Human Induced Pluripotent Stem Cells and Their Neural and Glial Derivatives

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential (MMP), mitochondrial production of reactive oxygen species (ROS), and mitochondrial DNA (mtDNA) copy number are often features of these processes. This report details a novel flow cytometry-based approach to measure multiple mitochondrial parameters in different cell types, including human induced pluripotent stem cells (iPSCs) and iPSC-derived neural and glial cells. This flow-based strategy uses live cells to measure mitochondrial volume, MMP, and ROS levels, as well as fixed cells to estimate components of the mitochondrial respiratory chain (MRC) and mtDNA-associated proteins such as mitochondrial transcription factor A (TFAM). 
By co-staining with fluorescent reporters, including MitoTracker Green (MTG), tetramethylrhodamine ethyl ester (TMRE), and MitoSox Red, changes in mitochondrial volume, MMP, and mitochondrial ROS can be quantified and related to mitochondrial content. Double staining with antibodies against MRC complex subunits and translocase of outer mitochondrial membrane 20 (TOMM20) permits the assessment of MRC subunit expression. As the amount of TFAM is proportional to mtDNA copy number, the measurement of TFAM per TOMM20 gives an indirect measurement of mtDNA per mitochondrial volume. The entire protocol can be carried out within 2-3 h. Importantly, these protocols allow the measurement of mitochondrial parameters, both at the total level and the specific level per mitochondrial volume, using flow cytometry.

This study reports a novel approach to measure multiple mitochondrial functional parameters based on flow cytometry and double staining with two fluorescent reporters or antibodies to detect changes in mitochondrial volume, mitochondrial membrane potential, reactive oxygen species level, mitochondrial respiratory chain composition, and mitochondrial DNA.

Mitochondria are important in the pathophysiology of many neurodegenerative diseases. Changes in mitochondrial volume, mitochondrial membrane potential ...

Flow Cytometry-Based Assays for Microglial Engulfment of Synaptic Materials

Quantification of Microglial Engulfment of Synaptic Material Using Flow Cytometry

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this process; however, currently available methods for identifying microglial engulfment of synapses, such as immunohistochemistry (IHC) and imaging, are laborious and time-intensive. To address this challenge, herein we present in vitro and in vivo* assays that allow fast and high-throughput quantification of microglial engulfment of synapses using flow cytometry.
In the in vivo* approach, we performed intracellular vGLUT1 staining following fresh cell isolation from adult mouse brains to quantify engulfment of vGLUT1+ synapses by microglia. In the in vitro synaptosome engulfment assay, we used freshly isolated cells from the adult mouse brain to quantify the engulfment of pHrodo&#160;Red-labeled synaptosomes by microglia. These protocols together provide a time-efficient approach to quantifying microglial engulfment of synapses and represent promising alternatives to labor-intensive image analysis-based methods. By streamlining the analysis, these assays can contribute to a better understanding of the role of microglia in synaptic refinement in different disease models.

Author Spotlight: Advancing Understanding Through Technological Innovations in Psychoneuroimmunology

Here we present two protocols to quantify microglial engulfment of vGLUT1-positive synapses and pHRodo&#160;Red-labeled crude synaptosomes using flow cytometry.

Microglia play a pivotal role in synaptic refinement in the brain. Analysis of microglial engulfment of synapses is essential for comprehending this ...

Flow Cytometry Protocols for Surface and Intracellular Antigen Analyses of Neural Cell Types

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