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&#8217;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 &#176;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 &#176;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 &#181;l) 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 &#176;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 &#181;m 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 &#8220;occupied&#8221; 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 &#181;l 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 &#181;M. 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="Figure 3" src="/files/ftp_upload/52241/52241fig3highres.jpg" width="700" /> 
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/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 &#181;l 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 &#181;l 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&#160;</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&#160;</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&#160;</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 &#176;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 &#181;l.</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 &#181;l of fixation buffer to 100 &#181;l 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 &#176;C.</li>
		<li>Discard/ decant the supernatant leaving approximately 100 &#181;l 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 &#181;l of permeabilization buffer to 100 &#181;l 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&#8217;s instructions.
		<ol>
			<li>Prepare 1 &#181;g of primary antibody in PBS at an appropriate dilution (total volume &#8804; 20 &#181;l).</li>
			<li>Add 5 &#181;l 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 &#181;l 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 &#181;l 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 &#181;l 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 &#181;l of flow buffer and analyze on the flow cytometer.</li>
</ol>
<img alt="Figure 4" src="/files/ftp_upload/52241/52241fig4highres.jpg" width="700" /> 
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/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 &#8804;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, microgl...

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="Figure 1" src="/files/ftp_upload/52241/52241fig1highres.jpg" width="600" /> 
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/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&#8217;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 &#8216;single file&#8217; 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&#8217;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&#8211;/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="Figure 2" src="/files/ftp_upload/52241/52241fig2highres.jpg" width="300" /> 
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/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&#8217;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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:456px;">DMEM/F12 (1:1) (Dulbecco&#39;s Modified Eagle Medium/Nutrient Mixture F-12)</td>
			<td style="width:207px;">Life Technologies</td>
			<td style="width:124px;">11330057</td>
			<td style="width:149px;">www.lifetechnologies.com</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DPBS without Ca2+ Mg2+</td>
			<td>Life Technologies</td>
			<td>14190169</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Fetal bovine serum, qualified, E.U.-approved, South America origin (FBS)</td>
			<td>Life Technologies</td>
			<td>10270-106</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MEM Non-essential amino acids (100 x)</td>
			<td>Life Technologies</td>
			<td>11140035</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">TrypLE Express</td>
			<td>Life Technologies</td>
			<td>12604013</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Trypan blue solution, 0.4%</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Paraformaldehyde</td>
			<td>Carl Roth</td>
			<td>335.3</td>
			<td>www.carlroth.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bovine serum albumin (BSA) Fraction V</td>
			<td>PAA Laboratories, Coelbe</td>
			<td>K41-001</td>
			<td>www.gelifesciences.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Tween-20 Detergent</td>
			<td>Calbiochem</td>
			<td>655205</td>
			<td>www.merckmillipore.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Carboxyfluorescein succinimidyl ester (CFSE)</td>
			<td>eBioscience</td>
			<td>65-0850-84</td>
			<td>www.ebioscience.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMSO</td>
			<td>AppliChem</td>
			<td>A1584</td>
			<td>www.applichem.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bottle top filters express plus 0.22 &#181;m, 250 ml</td>
			<td>Millipore</td>
			<td>SCGPU02RE</td>
			<td>www.millipore.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell culture treated flasks (T 25)</td>
			<td>NUNC</td>
			<td>156367</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cell culture treated flasks (T 75)</td>
			<td>NUNC</td>
			<td>156499</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Conical tubes (15 ml)</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Conical tubes (50 ml)</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pasteur pipet, glass (150 mm)</td>
			<td>STEIN Labortechnik, Remchingen</td>
			<td>S03710150</td>
			<td>www.stein-labortechnik.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet tips (0.1-10 &#181;l)</td>
			<td>Corning</td>
			<td>4125</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet tips (1-200 &#181;l)</td>
			<td>Corning</td>
			<td>4126</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet tips (100-1000 &#181;l)</td>
			<td>Corning</td>
			<td>4129</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Serological pipets, 5 ml</td>
			<td>Corning</td>
			<td>4051</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Serological pipets, 10 ml</td>
			<td>Corning</td>
			<td>4101</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Serological pipets, 25 ml</td>
			<td>Corning</td>
			<td>4251</td>
			<td>www.corning.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge tubes (0.5 ml)</td>
			<td>Sarstedt</td>
			<td>72,699</td>
			<td>www.sarstedt.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge tubes (1.5 ml)</td>
			<td>Greiner Bio-One</td>
			<td>616201</td>
			<td>www.greinerbioone.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge tubes (2.0 ml)</td>
			<td>Sarstedt</td>
			<td>72,695,500</td>
			<td>www.sarstedt.com</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human CD24 APC monoclonal antibody</td>
			<td>eBioscience</td>
			<td>17-0247-42</td>
			<td>www.ebioscience.com 
			Working dilution 1:50</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-Human CD54 PE monoclonal antibody</td>
			<td>eBioscience</td>
			<td>12-0549-42</td>
			<td>www.ebioscience.com 
			Working dilution 1:50</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:456px;">Neuronal Class III &#946;-Tubulin (Tuj1) polyclonal antibody</td>
			<td>Covance</td>
			<td>PRB-435P</td>
			<td>www.covance.com 
			Working dilution 1:2000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Alexa Fluor 488 Donkey anti Rabbit</td>
			<td>Life Technologies</td>
			<td>A21206</td>
			<td>www.lifetechnologies.com 
			Working dilution 1:2000</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Zenon&#174; Fluorescein Rabbit IgG Labeling Kit</td>
			<td>Life Technologies</td>
			<td>Z-25342</td>
			<td>www.lifetechnologies.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Neubauer-Improved counting chamber</td>
			<td>Marienfeld</td>
			<td>0640010</td>
			<td>www.marienfeld-superior.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vortex</td>
			<td>Scientific Industries</td>
			<td>G560E</td>
			<td>www.scientificindustries.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Thermomixer comfort</td>
			<td>Eppendorf</td>
			<td>5355 000.001</td>
			<td>www.eppendorf.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Accuri C6 flow cytometer</td>
			<td>Becton Dickinson (BD)</td>
			<td align="right">653118</td>
			<td>www.bdbiosciences.com/instruments/accuri</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microcentrifuge refrigerated, PerfectSpin 24 R</td>
			<td>Peqlab</td>
			<td>91-PSPIN-24R</td>
			<td>www.peqlab.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Orbital shaker, Unimax 1010</td>
			<td>Heidolph</td>
			<td>543-12310-00</td>
			<td>www.heidolph-instruments.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Centrifuge refrigerated, Rotanta 96 RC</td>
			<td>Hettich</td>
			<td>4480-50</td>
			<td>www.hettichlab.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Class II Biological safety cabinet Safe 2020</td>
			<td>Thermo Scientific</td>
			<td>51026640</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CO2 Incubator, Heracell 240i</td>
			<td>Thermo Scientific</td>
			<td align="right">51026331</td>
			<td>www.thermoscientific.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Vacuum system, Vacusafe comfort</td>
			<td>Integra Biosciences</td>
			<td align="right">158320</td>
			<td>www.integra-biosciences.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Microscope, Axiovert 40 CFL</td>
			<td>Zeiss</td>
			<td align="right">451212</td>
			<td>www.zeiss.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Pipet controller, accu-jet pro</td>
			<td>Brand</td>
			<td align="right">26303</td>
			<td>www.brand.de</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micropipet, Pipetman neo P20N (2-20 &#181;l)</td>
			<td>Gilson</td>
			<td>F144563</td>
			<td>www.gilson.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micropipet, Pipetman neo P200N (20-200 &#181;l)</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td>www.gilson.com</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micropipet, Pipetman neo P1000N (100-1000 &#181;l)</td>
			<td>Gilson</td>
			<td>F144566</td>
			<td>www.gilson.com</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 label...

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

Research

JoVE Journal

Neuroscience

51.8K 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

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

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 ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Traditional methods of immunohistochemistry (IHC) following tissue fixation allow visualization of various cell types. These typically proceed with the application of antibodies to bind antigens and identify cells with characteristics that are a function of the inherent biology and development. Adult hippocampal neurogenesis is a sequential process wherein a quiescent neural stem cell can become activated and proceed through stages of proliferation, differentiation, maturation and functional integration. Each phase is distinct with a characteristic morphology and upregulation of genes. Identification of these phases is important to understand the regulatory mechanisms at play and any alterations in this process that underlie the pathophysiology of debilitating disorders. Our heat-induced antigen retrieval approach improves the intensity of the signal that is detected and allows correct identification of the progenitor cell type. As discussed in this paper, it especially allows us to circumvent current problems in detection of certain progenitor cell types.

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Traditional  methods of immunohistochemistry (IHC) following tissue fixation allow visualization  of various cell types. These typically proceed with the ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Enumeration of Neural Stem Cells Using Clonal Assays

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. NSCs and neural progenitors (NPs) are commonly cultured in vitro as neurospheres. This protocol describes in detail how to determine the NSC frequency in a given cell population under clonal conditions. The protocol begins with the seeding of the cells at a density that allows for the generation of clonal neurospheres. The neurospheres are then transferred to chambered coverslips and differentiated under clonal conditions in conditioned medium, which maximizes the differentiation potential of the neurospheres. Finally, the NSC frequency is calculated based on neurosphere formation and multipotency capabilities. Utilities of this protocol include the evaluation of candidate NSC markers, purification of NSCs, and the ability to distinguish NSCs from NPs. This method takes 13 days to perform, which is much shorter than current methods to enumerate NSC frequency.

Neural stem cells (NSCs) refer to cells&#160;which can self-renew and differentiate into the three neural lineages. Here, we describe a protocol to determine NSC frequency in a given cell population using neurosphere formation and differentiation under clonal conditions.

Neural stem cells (NSCs) have the ability to self-renew and generate the three major neural lineages &#8212; astrocytes, neurons and oligodendrocytes. ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications

Presented here is a protocol that combines an in vitro neural-endothelial co-culture system and metabolic incorporation of sialoglycan with bioorthogonal functional groups to expand primary neural stem and progenitor cells and label their surface sialoglycoproteins for imaging or mass-spectrometry analysis of cell surface markers.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized ...