Name: Video: Isolating Fluorescently Labeled Neurons from a Murine Brain - Concept
Uploaded: 2024-08-30T13:23:56.000+00:00
Duration: 1 min 20 s
Description: 41 Views. Source: Paul, A., et al., Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq). J. Vis. Exp. (2018) This video demonstrates a method to isolate fluorescently labeled neurons from a mouse brain.

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All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Manual Sorting of Fluorescently Labeled Mouse Neurons
<ol>
	<li>Pull glass microcapillaries (see Table of Materials) to 10&#8211;15 &#181;m exit diameter using a capillary puller with the following settings: heat: 508, pull: blank, vel: blank, time: blank.</li>
	<li>Attach 120&#8211;150 cm flexible silicone tubing (~0.8 mm inside diameter) to a 0.2 &#181;m polyvinylidene difluoride (PVDF) membrane syringe filter and a two-way tubing valve using suitable tubing connectors.</li>
	<li>Prepare 500 mL of chilled (4 &#176;C) artificial cerebrospinal fluid (ACSF, Table 1), and oxygenate by bubbling 5% carbon dioxide balanced oxygen through an airstone for 15 min or until the solution clears completely.</li>
	<li>Prepare 100 mL of ACSF with a cocktail of activity blockers in a 150 mL beaker (Table 1).</li>
	<li>Prepare 100 mL of ACSF with 1 mg/mL Streptococcus fraction IV protease in a 150 mL beaker (Table 1).</li>
	<li>Prepare 100 mL of ACSF with 1% fetal bovine serum (FBS) solution in a 150 mL beaker and oxygenate with 5% carbon dioxide balanced oxygen through an airstone.</li>
	<li>Dissect the brain from an euthanized mouse by opening the cranium with a small scissor and extracting the fresh brain using fine forceps without damaging the cortex. 
	NOTE: Mice of any strain, age, and gender can be used as desired. Do not freeze the brain or perform manual sorting on mice injected with viruses or other hazardous/infectious agents.</li>
	<li>Keep the brain in chilled and oxygenated ACSF, leaving an airstone attached to 5% carbon dioxide balance oxygen during the entire duration of the sectioning.</li>
	<li>Position the brain on the vibratome chuck and cut coronal or sagittal sections at 300 &#181;m thickness. Collect as many sections as needed to obtain a minimum of ~10&#8211;50 labeled cells up to several hundred cells. Put slices on a cotton meshed slice holder, placed inside a beaker so they are bathed in oxygenated ACSF.</li>
	<li>Move fresh slices into a beaker containing ACSF with activity blockers and block for 15&#8211;20 min at room temperature. Keep bubbling oxygen using airstone.</li>
	<li>Move the slices to a beaker containing ACSF with the protease solution to perform mild digestion at room temperature: 20&#8211;30 min for P4-14 animals or up to 45&#8211;60 min for P28-56 animals. Keep bubbling oxygen.</li>
	<li>Wash out ACSF with protease by moving slices back to the beaker containing ACSF with activity blockers solution for 5&#8211;10 min at room temperature. Keep bubbling oxygen.</li>
	<li>Move individual sections to a 100 mm Petri dish containing ACSF with 1% FBS at room temperature. Under a fluorescent dissection scope, microdissect areas and layers of interest having a minimum of 10&#8211;50 cells (up to a few hundred). Perform the microdissection with a pair of fine forceps or by attaching 22&#8211;28 G injection needles onto wooden holders (skewers).</li>
	<li>Using a Pasteur pipette, move the microdissected pieces to a 2 mL microfuge tube containing ~0.8 mL of 1% FBS in ACSF solution.</li>
	<li>Triturate the dissected tissue in the microfuge tube at room temperature. Make three long-stemmed Pasteur pipettes with decreasing exit diameters by rolling them over an open flame. Perform ~10 strokes with each pipette, starting with the biggest and ending with the smallest.</li>
	<li>Dispense the dissociated cells into a 100 mm Petri dish containing oxygenated ACSF (Petri dish can be thinly coated with 5 mm of 1% agar or clear silicone compound at 1:10 ratio, if desired). Keep at room temperature.</li>
	<li>Wait ~5-7 min for the cells to gradually settle, then observe the GFP/RFP signal under a dissection microscope. 
	NOTE: Single cells, debris, and small clumps will be visible in bright-field (BF).</li>
	<li>Pick 10&#8211;15 GFP/RFP cells at a time using a capillary pipette (from step 1.1) attached to flexible silicone tubing (0.4&#8211;0.8 mm inner diameter) and dispense the cells into a 100 mm Petri dish containing fresh oxygenated ACSF.</li>
	<li>By blocking the end of the tubing valve with the tongue, position the capillary close to the cell of interest. Capture cells using capillary action upon relieving the block, and quickly block again to prevent excess fluid from entering the pipette. Dispense the cells onto a 100 mm Petri dish with fresh oxygenated ACSF by gently blowing, while observing the capillary tip under fluorescence optics of the dissection microscope. Aim to collect ~100&#8211;150 cells total.</li>
	<li>Repeat step 1.19 once or twice more (depending on debris) and transfer a total of ~50&#8211;75 cells to a new 100 mm Petri dish, making sure that contaminants such as debris are minimal in bright-field differential interference contrast (DIC) optics.</li>
</ol>
Table 1: List of solutions and buffers.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Item</td>
			<td>Concentration</td>
			<td>Amount (&#181;L)</td>
		</tr>
		<tr>
			<td>Sample Collection buffer</td>
			<td>Recombinant ribonuclease inhibitor</td>
			<td></td>
			<td>55</td>
		</tr>
		<tr>
			<td></td>
			<td>RCC</td>
			<td>1:50K diluted</td>
			<td>110</td>
		</tr>
		<tr>
			<td></td>
			<td>Nuclease free water</td>
			<td></td>
			<td>605</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Aliquot 43.75 &#181;L of above into 16 tubes (2 strips of 8; 200 &#181;L PCR tubes); add following per tube</td>
		</tr>
		<tr>
			<td></td>
			<td>UMI-primers (e.g. N10B1-N10B16)</td>
			<td>1 ng/&#181;L</td>
			<td>6.25&#160; &#181;L/tube</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Final volume in each tube 50 &#181;L (each tube can be split in 25 &#181;L aliquots and frozen at -80 &#176;C)</td>
		</tr>
		<tr>
			<td>Solutions:</td>
			<td>Item</td>
			<td>Concentration</td>
			<td>Amount</td>
		</tr>
		<tr>
			<td>CSF: to make 5 L dissolve</td>
			<td>NaCl</td>
			<td>126 mM</td>
			<td>36.8 g</td>
		</tr>
		<tr>
			<td></td>
			<td>KCl</td>
			<td>3 mM</td>
			<td>1.15 g</td>
		</tr>
		<tr>
			<td></td>
			<td>NaH2PO4</td>
			<td>1.25 mM</td>
			<td>0.75 g</td>
		</tr>
		<tr>
			<td></td>
			<td>NaHCO3</td>
			<td>20 mM</td>
			<td>8.4 g</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">To 500 mL of ACSF, bubble oxygen for 10-15 min then add following fresh each time:</td>
		</tr>
		<tr>
			<td></td>
			<td>D-glucose</td>
			<td>20 mM</td>
			<td>1.8 g</td>
		</tr>
		<tr>
			<td></td>
			<td>MgSO4</td>
			<td>2 mM</td>
			<td>0.5 mL from 4 M stock</td>
		</tr>
		<tr>
			<td></td>
			<td>CaCl2</td>
			<td>2 mM</td>
			<td>0.5 mL from 4 M stock</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="3">Keep ACSF oxygenated through out</td>
		</tr>
		<tr>
			<td>Solutions:</td>
			<td>Item</td>
			<td colspan="2">Concentration</td>
		</tr>
		<tr>
			<td colspan="4">Activity blocker cocktail: make a 100x stock</td>
		</tr>
		<tr>
			<td></td>
			<td>To 100 mL ACSF add</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>APV</td>
			<td>0.05 mM</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>CNQX</td>
			<td>0.02 mM</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>TTX</td>
			<td colspan="2">0.0001 mM (0.1 &#181;M)</td>
		</tr>
		<tr>
			<td>Solutions:</td>
			<td>Item</td>
			<td>Amount</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease soltion (100 mL)</td>
			<td>Protease from Streptomyces griseus</td>
			<td>100 mg</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Fetal bovine serum: commercial source, aliquot in 500 &#181;L for each use.</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DL-AP5</td>
			<td>Tocris</td>
			<td>Cat# 0105</td>
			<td></td>
		</tr>
		<tr>
			<td>CNQX</td>
			<td>Tocris</td>
			<td>Cat# 1045</td>
			<td></td>
		</tr>
		<tr>
			<td>TTX</td>
			<td>Tocris</td>
			<td>Cat# 1078</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease from Streptomyces griseus</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Airgas</td>
			<td>Cat# UN3156</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# 761036</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope with fluorescence and bright field illumination with DIC optics. (Leica model MZ-16F).</td>
			<td>Leica</td>
			<td>Model MZ-16F</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass microcapillary: Borosilicate capillary tubes 500/pk. OD=1 mm, ID=0.58 mm, wall=0.21 mm, Length=150 mm.</td>
			<td>Warner instruments</td>
			<td>Model GC100-15, Order# 30-0017</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary pipette puller</td>
			<td>Sutter Instruments Co</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Thermo Microm</td>
			<td>HM 650V</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome tissue cooling unit</td>
			<td>Thermo Microm</td>
			<td>CU 65</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating fluorescently labeled neurons from a murine brain

Source: Paul, A., et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/58690">Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq).</a> J. Vis. Exp. (2018)
This video demonstrates a method to isolate fluorescently labeled neurons from a mouse brain.

Isolating Fluorescently Labeled Neurons from a Murine Brain

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Manual Sorting of Fluorescently ...

Take a transgenic mouse cortex containing fluorescent protein-expressing neurons.
Secure it on a sample holder.
Mount the holder on the vibratome tray containing oxygenated aCSF to maintain the tissue&#39;s physiological conditions.
Using set parameters, obtain coronal slices.
Place the slices in a meshed holder containing oxygenated aCSF and activity blockers to stabilize them.
Treat the slices with proteases to digest the extracellular matrix and loosen cells.
Wash with aCSF to remove the proteases.
Transfer the slices to a culture plate with aCSF and serum.
Under a fluorescence microscope, dissect slice regions containing fluorescent neurons.
Transfer the dissected fragments to a tube containing aCSF and serum.
Mechanically dissociate the tissue to form a single-cell suspension. Transfer the cells to a culture plate containing aCSF.
Under a fluorescence microscope, use a capillary pipette to collect fluorescent neurons.
Dispense these neurons into a culture plate containing aCSF for further analysis.

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Neuronal Culture Techniques

Primary Neuronal Culture

41 Views. Source: Paul, A., et al., Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq). J. Vis. Exp. (2018) This video demonstrates a method to isolate fluorescently labeled neurons from a mouse brain. 

Video: Isolating Fluorescently Labeled Neurons from a Murine Brain - Concept

Fluorescence-activated Cell Sorting for Purification of Plasmacytoid Dendritic Cells from the Mouse Bone Marrow

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method enables researchers to better understand the characteristics of a single cell population without the influence of other cells. Compared to other methods of cell enrichment, such as magnetic-activated cell sorting (MCS), FACS is more flexible and accurate for cell separation due to the ability of phenotype detection by flow cytometry. In addition, FACS is usually capable of separating multiple cell populations simultaneously, which improves the efficiency and diversity of experiments. Although FACS has some limitations, it has been broadly used to purify cells for functional studies in both in vitro and in vivo settings. Here we report a protocol using fluorescence-activated cell sorting to isolate a very rare population of immune cells, plasmacytoid dendritic cells (pDC), with high purity from the bone marrow of lupus-prone mice for in vitro functional studies of pDC.

We report a protocol using fluorescence-activated cell sorting to isolate plasmacytoid dendritic cells (pDC) with high purity from the bone marrow of lupus-prone mice for functional studies of pDC.

Fluorescence-activated cell sorting (FACS) is a technique to purify specific cell populations based on phenotypes detected by flow cytometry. This method ...

JoVE Journal

Immunology and Infection

Generation of Large Numbers of Myeloid Progenitors and Dendritic Cell Precursors from Murine Bone Marrow Using a Novel Cell Sorting Strategy

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have recently been recognized to be more heterogeneous than previously appreciated. These cultures routinely contain moDC as well monocyte-derived macrophages (moMac), and even some less developed cells such as monocytes. The goal of this protocol is to provide a consistent method for identification and separation of the many cell types present in these cultures as they develop, so that their specific functions may be further investigated. The sorting strategy presented here separates cells first into four populations based on expression of Ly6C and CD115, both of which are expressed transiently by cells as they develop in GM-CSF-driven culture. These four populations include Common myeloid progenitors or CMP (Ly6C-, CD115-), granulocyte/macrophage progenitors or GMP (Ly6C+, CD115-), monocytes (Ly6C+, CD115+), and monocyte-derived macrophages or moMac (Ly6C-, CD115+). CD11c is also added to the sorting strategy to distinguish two populations within the Ly6C-, CD115- population: CMP (CD11c-) and moDC (CD11c+). Finally, two populations may be further distinguished within the Ly6C-, CD115+ population based on the level of MHC class II expression. MoMacs express lower levels of MHC class II, while a monocyte-derived DC precursor (moDP) expresses higher MHC class II. This method allows for the reliable isolation of several developmentally distinct populations in numbers sufficient for a variety of functional and developmental analyses. We highlight one such functional readout, the differential responses of these cell types to stimulation with Pathogen-Associated Molecular Patterns (PAMPs).

Here we provide a method for identifying and isolating large numbers of GM-CSF driven myeloid cells using high speed cell sorting. Five distinct populations (Common myeloid progenitors, granulocyte/macrophage progenitors, monocytes, monocyte-derived macrophages, and monocyte-derived DCs) can be identified based on Ly6C and CD115 expression.

Cultures of monocyte-derived dendritic cells (moDC) generated from mouse bone marrow using Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) have ...

Primary Cell Culture of Purified GABAergic or Glutamatergic Neurons Established through Fluorescence-activated Cell Sorting

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can be cultured in defined media for 16 days in vitro and are amenable to any analyses typically performed on dissociated cultures, including electrophysiological, morphological and survival analyses. The major advantage of these cultures is that specific cell types can be selectively studied in the absence of complex external influences, such as those arising from glial cells or other neuron types. When planning experiments with purified cells, however, it is important to note that neurons strongly depend on glia-conditioned media for their growth and survival. In addition, glutamatergic neurons further depend on glia-secreted factors for the establishment of synaptic transmission. We therefore also describe a method for co-culturing neurons and glial cells in a non-contact arrangement. Using these methods, we have identified major differences between the development of GABAergic and glutamatergic neuronal networks. Thus, studying cultures of purified neurons has great potential for furthering our understanding of how the nervous system develops and functions. Moreover, purified cultures may be useful for investigating the direct action of pharmacological agents, growth factors or for exploring the consequences of genetic manipulations on specific cell types. As more and more transgenic animals become available, labeling additional specific cell types of interest, we expect that the protocols described here will grow in their applicability and potential.

This protocol describes a cell sorting based method for the purification and culture of fluorescent GABAergic or glutamatergic neurons from the neocortex and hippocampus of postnatal mice or rats.

The overall goal of this protocol is to generate purified neuronal cultures derived from either GABAergic or glutamatergic neurons. Purified neurons can ...