isolation of multiple cell types from a mouse brain by mechanical homogenization

Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

For tissue homogenization, add 3 milliliters of prechilled HBSS to the prechilled glass mortar of a Dounce tissue grinder, and transfer the half of one of the harvested brain to the mortar. Gently macerate the tissue with 10 strokes of pestle A, followed by 10 strokes of pestle B, and transfer the homogenized mixture into a new 15-milliliter conical tube.
Fill the tube to a final volume of 10 milliliters with prechilled HBSS for centrifugation and resuspend the pellet in 7 milliliters of fresh HBSS with vortexing before holding the sample on ice. For debris removal, add 3 milliliters of prechilled isotonic solution to each digested or homogenized sample, and gently vortex the samples to make sure they are homogeneously mixed.
Next, centrifuge the samples and carefully remove the whitish disk of debris and myelin floating at the surface of the solution. When the debris has been discarded, collect all but the last 100 microliters of the supernatant from each tube without disturbing the pellets. Resuspend each pellet in 1 milliliter of FACS/BL solution for transfer into individual 1.5-milliliter microcentrifuge tubes.
After the centrifugation with the particle solution, it&#39;s important to remove the debris disk and the supernatant very carefully, without dislodging the cell pellet, to avoid sample loss.
After centrifugation, carefully aspirate the supernatant from each tube and resuspend the pellets in 350 microliters of fresh FACS/BL per tube.

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Disclaimer: &#160;All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical homogenization of the brain and spinal cord
NOTE: Volumes described in this section are enough for the homogenization of one-half brain or spinal cord.
<ol>
	<li>Pre-chill the glass mortar of the Dounce tissue grinder (Table of Materials) set on ice.</li>
	<li>Add 3 mL of pre-chilled 1x Hank&#39;s balanced salt solution (HBSS) to the mortar.</li>
	<li>Transfer the tissue (brain or spinal cord) from the well of the 6-well plate into the glass mortar, making sure it is dipped in 1x HBSS and sits at the bottom of the mortar.</li>
	<li>Gently smash the tissue with 10 strokes of pestle A followed by 10 strokes of pestle B. Transfer the homogenized mix into a new 15 mL conical tube.</li>
	<li>Fill the tube to a final volume of 10 mL by using pre-chilled 1x HBSS and centrifuge for 10 min at 320 x g at 4 &#176;C.</li>
	<li>Aspirate the supernatant and add ice-cold 1x HBSS to each tube up to a final volume of 7 mL and gently resuspend the pellet by vortexing.</li>
</ol>
2. Debris removal 
NOTE: Removal of debris, composed mainly of undigested tissue and myelin sheaths, is a critical step to allow efficient staining of the tissue homogenate for subsequent flow cytometric analyses.
<ol>
	<li>Filter each sample through a 70 &#181;m cell strainer to remove any undigested tissue chunks. This step is particularly important when working with spinal cord tissues, as these samples are more likely to contain undigested nerve fragments or meninges that could affect the subsequent steps.</li>
	<li>Make sure that the final volume is 7 mL in each sample tube. If not, fill with ice-cold 1x HBSS up to 7 mL.</li>
	<li>Add 3 mL of pre-chilled isotonic Percoll solution (IPS) to each sample to make a final volume of 10 mL of a solution containing density gradient medium at a 30% final concentration. Gently vortex the samples to ensure they are homogeneously mixed.</li>
	<li>Centrifuge samples for 15 min at 700 x g at 18 &#176;C, making sure to set the acceleration of the centrifuge to 7 and the brake to 0. 
	NOTE: Centrifugation should take approximately 30 min.</li>
	<li>Delicately remove the samples from the centrifuge. 
	NOTE: A whitish disk composed of debris and myelin should be visible floating on the surface of the solution. A pellet (containing the cells of interest) should be visible at the bottom of the tube.</li>
	<li>Carefully aspirate all the whitish disk of debris and then the rest of the supernatant, making sure not to dislodge the pellet. Leave about 100 &#181;L of solution on top of the cell pellet to avoid the risk of inadvertently dislodging it.</li>
	<li>Add 1 mL of flow cytometry (FACS) blocking (BL) solution, resuspend the pellet by pipetting up and down with a 1000 &#181;L pipette tip, and transfer samples to 1.5 mL tubes.</li>
	<li>Centrifuge for 5 min at 450 x g at room temperature (RT).</li>
	<li>Gently aspirate the supernatant and resuspend the pellet in the appropriate buffer compatible with downstream analyses</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10X HBSS (Calcium, Magnesium chloride, and Magnesium Sulfate-free)</td>
			<td>Gibco</td>
			<td>14185-052</td>
			<td></td>
		</tr>
		<tr>
			<td>70 mm Cell Strainer</td>
			<td>Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes (15 mL)</td>
			<td>CellTreat</td>
			<td>229411</td>
			<td></td>
		</tr>
		<tr>
			<td>Conical Tubes (50 mL)</td>
			<td>CellTreat</td>
			<td>229422</td>
			<td></td>
		</tr>
		<tr>
			<td>Dounce Tissue Grinder set (Includes Mortar as well as Pestles A and B)</td>
			<td>Sigma-Aldrich</td>
			<td>D9063-1SET</td>
			<td></td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>GE Healthcare</td>
			<td>10266569</td>
			<td>sold as not sterile reagent</td>
		</tr>
		<tr>
			<td>Percoll</td>
			<td>Sigma</td>
			<td>65455529</td>
			<td>sterile reagent (to be used for applications requiring sterility)</td>
		</tr>
		<tr>
			<td>Percoll PLUS</td>
			<td>Sigma</td>
			<td>GE17-5445-01</td>
			<td>reagent containing very low traces of endotoxin</td>
		</tr>
	</tbody>
</table>

Source: Molina Estevez, F. J., et al. <a href="https://app.jove.com/v/60335">Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods.</a> J. Vis. Exp. (2019).
This video demonstrates the isolation of multiple types of cells from mouse brain tissue. Tissue is first mechanically sheared using a tissue grinder in a salt solution to maintain an osmotic balance. The tissue homogenate is then subjected to density gradient centrifugation to remove cellular debris. Cells are then resuspended in a solution for further analysis.

Disclaimer: &#160;All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Mechanical ...

Begin by placing the tissue grinder components on ice. This device includes a mortar and two pestles of different diameters.
Pour a chilled salt buffer into the mortar and then add mouse brain tissue.
The buffer maintains osmotic balance, preserving cell structure and function.
Grind the tissue with multiple gentle strokes using the smaller diameter pestle, which mechanically shears the tissue into smaller pieces.
Next, stroke with the larger diameter pestle, facilitating further breakdown of tissue into its component cells.
Transfer the mixture to a tube and centrifuge.
Remove the supernatant.
Add chilled salt buffer, and vortex to break the cell clumps and the myelin.
Next, add a density gradient medium and centrifuge.
Due to differences in shape and mass, cells will settle in the lower layer, while debris and myelin accumulate in the top layer.
Discard the supernatant.
Add a suitable solution to obtain a multiple-cell suspension for further use.

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Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization

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Isolation of Multiple Cell Types from a Mouse Brain by Mechanical Homogenization