Name: Video: Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection - Concept
Uploaded: 2023-07-31T12:11:56.000+00:00
Duration: 1 min 57 s
Description: 907 Views. Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019) In this video, we demonstrate the isolation of monocytes from whole human blood by immunomagnetic negative separation. The isolated monocytes can be used for further downstream analysis.

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All procedures involving human participants have been performed in compliance with the institutional, national, and international guidelines for human welfare and have been reviewed by the local institutional review board.
1. Isolation of primary human monocytes by immunomagnetic negative selection
<ol>
	<li>Collect 40 mL of fresh, human whole blood (from either an HIV+ patient or healthy control) in four 10 mL ethylenediaminetetraacetic acid (EDTA) vacuum tubes (10 mL of blood per tube). Using sterile techniques under a biosafety cabinet, transfer all 40 mL of blood into one 50 mL conical propylene tube.</li>
	<li>Following the manufacturer&#39;s protocol for the selected human monocyte isolation kit (Table of Materials), add 2 mL of monocyte isolation cocktail, provided in the kit, to the tube of blood. Vortex magnetic beads, also provided in the kit, for 30 s, and add 2 mL to the tube of blood.
	<ol>
		<li>If less than 40 mL of blood is available, scale down the reagents added. To mix the solution, pipette up and down with a plastic 25 mL serological pipette and incubate for 5 min at room temperature (RT).</li>
	</ol>
	</li>
	<li>Separate the blood mixture equally into four 50 mL tubes and add 30 mL of sterile phosphate-buffered saline (PBS) containing 1 mM EDTA to each tube. Mix by pipetting up and down with a plastic 25 mL serological pipette.</li>
	<li>Place the tubes in magnet holders for 10 min to remove the antibody-conjugated magnetic beads. Use four magnet holders simultaneously, one for each tube, to allow consistent incubation and isolation times for each blood sample.</li>
	<li>Draw up the contents from the center of each tube, using a pipette, while they are still in the magnet holders. Be careful not to draw up red blood cells (no more than 10% of the 10 mL starting volume), and place the contents into one of four new 50 mL tubes.</li>
	<li>Add 500 &#181;L of vortexed magnetic beads to each 50 mL tube. Pipette up and down with a 25 mL pipette and incubate at RT for 5 min. Then, place the tubes into magnet holders for 5 min.</li>
	<li>Carefully transfer the contents from the center of each tube while still in magnet holders into one of four new 50 mL tubes. Directly place each new 50 mL tube in the magnet holders for 5 min.</li>
	<li>Carefully transfer contents from the center of each tube into one of four new 50 mL tubes. Spin all new 50 mL tubes at 300 x g for 5 min. Aspirate the supernatant and resuspend all four cell pellets in a total of 10 mL of sterile PBS.</li>
	<li>Count cells by trypan blue exclusion using a hemocytometer. 
	NOTE: 8-20 x 106 cells are generally obtained from 40 mL of whole blood.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5M EDTA</td>
			<td>Invitrogen</td>
			<td>AM9260G</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Plastic Blood Collection Tubes with K2EDTA</td>
			<td>BD Biosciences</td>
			<td>366643</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy 50 EasySep Magnet</td>
			<td>StemCell Technologies</td>
			<td>18002</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy Sep Direct Human Monocyte Isolation Kit</td>
			<td>StemCell Technologies</td>
			<td>19669</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>20012027</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 with L-Glutamine</td>
			<td>Corning</td>
			<td>10040CVMP</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of monocytes from whole blood by immunomagnetic negative selection

Source: Plaisance-Bonstaff, K., et al. <a href="https://app.jove.com/v/59967">Isolation, Transfection, and Culture of Primary Human Monocytes.</a> J. Vis. Exp. (2019)
In this video, we demonstrate the isolation of monocytes from whole human blood by immunomagnetic negative separation. The isolated monocytes can be used for further downstream analysis.

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019)
In this video, we demonstrate ...

To isolate monocytes, a type of white blood cell, from whole blood by immunomagnetic negative selection, obtain anticoagulant-treated human blood in a tube. Add an antibody cocktail solution containing Fc receptor-blocking antibodies and bispecific tetrameric antibody complexes.
Each antibody complex comprises two monoclonal antibodies, with one antibody specific for cell surface antigens on the non-monocyte cells, including white and red blood cells, and the other antibody specific for dextran.
Add a dextran-coated magnetic bead solution. Incubate. The Fc receptor-blocking antibody binds to the monocytes&#39; Fc receptor, preventing non-specific binding of monoclonal antibodies.
The monoclonal antibodies in antibody complexes recognize and bind to specific cell surface antigens on the non-monocyte cells, leaving the monocytes unlabeled. The dextran-coated magnetic beads bind to the antibody complexes&#39; anti-dextran antibody, crosslinking the non-monocyte cells to the beads.
Place the tube in a magnetic holder. Under the influence of a high-gradient magnetic field, the magnetic bead-crosslinked cells bind to the tube walls, leaving the monocytes in the center of the tube.
Pipette the monocyte-containing solution; transfer to a fresh tube. Add magnetic beads, crosslinking remaining unwanted cells. Place under a magnetic field&#39;s influence, enriching the monocyte population.
Transfer the monocyte suspension to a fresh tube. Centrifuge the suspension. Resuspend the monocytes in a buffer for further analysis.

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907 Views. Source: Plaisance-Bonstaff, K., et al. Isolation, Transfection, and Culture of Primary Human Monocytes. J. Vis. Exp. (2019) In this video, we demonstrate the isolation of monocytes from whole human blood by immunomagnetic negative separation. The isolated monocytes can be used for further downstream analysis. 

Video: Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection - Concept

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Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

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