Name: 视频: 通过免疫磁性阴性选择从全血中分离单核细胞 - 概念
Uploaded: 2023-07-31T12:11:56.000+00:00
Duration: 1 min 57 s
Description: 907 次观看. 来源:Plaisance-Bonstaff， K. 等人。原代人单核细胞的分离、转染和培养。J. Vis. Exp. （2019 年） 在本视频中，我们演示了通过免疫磁阴性分离从全人血液中分离单核细胞。分离的单核细胞可用于进一步的下游分析。

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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 次观看. 来源:Plaisance-Bonstaff， K. 等人。原代人单核细胞的分离、转染和培养。J. Vis. Exp. （2019 年） 在本视频中，我们演示了通过免疫磁阴性分离从全人血液中分离单核细胞。分离的单核细胞可用于进一步的下游分析。 

视频: 通过免疫磁性阴性选择从全血中分离单核细胞 - 概念

A Simple Flow Cytometric Method to Measure Glucose Uptake and Glucose Transporter Expression for Monocyte Subpopulations in Whole Blood

Monocytes are innate immune cells that can be activated by pathogens and inflammation associated with certain chronic inflammatory diseases. Activation of monocytes induces effector functions and a concomitant shift from oxidative to glycolytic metabolism that is accompanied by increased glucose transporter expression. This increased glycolytic metabolism is also observed for trained immunity of monocytes, a form of innate immunological memory. Although in vitro protocols examining glucose transporter expression and glucose uptake by monocytes have been described, none have been examined by multi-parametric flow cytometry in whole blood. We describe a multi-parametric flow cytometric protocol for the measurement of fluorescent glucose analog 2-NBDG uptake in whole blood by total monocytes and the classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++) monocyte subpopulations. This method can be used to examine glucose transporter expression and glucose uptake for total monocytes and monocyte subpopulations during homeostasis and inflammatory disease, and can be easily modified to examine glucose uptake for other leukocytes and leukocyte subpopulations within blood.

Monocytes are integral components of the human innate immune system that rely on glycolytic metabolism when activated. We describe a flow cytometry protocol to measure glucose transporter expression and glucose uptake by total monocytes and monocyte subpopulations in fresh whole blood.

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Quantification of Monocyte Transmigration and Foam Cell Formation from Individuals with Chronic Inflammatory Conditions

Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the transmigration of innate immune monocytes to inflammatory sites of deposited lipid called fatty streaks, which are present in arterial walls of medium to large arteries. The key pathogenic feature of lesions at this early stage of atherosclerosis is the maturation of monocytes which migrate into arteries to form foam cells or lipid-laden macrophages. Considerable evidence supports the hypothesis that risk of atherosclerosis is increased by chronic inflammatory conditions accompanying diseases such as rheumatoid arthritis and HIV, as well as general ageing, and that this risk is predicted by monocyte activation. While mouse models provide a good platform to investigate the role of monocytes in atherogenesis in vivo, they require genetic alteration of natural cholesterol metabolism and drastic alteration of normal mouse diets, and have limited suitability for the study of atherogenic influences of human comorbid diseases. This motivated us to develop a human in vitro model to measure the atherogenic potential of monocytes isolated from individuals with defined disease states. Currently, human in vitro models are limiting in that they evaluate monocyte transmigration and foam cell formation in isolation. Here we describe a protocol in which monocytes isolated from patient blood transmigrate across human endothelial cells into a type 1 collagen matrix, and their propensity to mature into foam cells in the presence or absence of exogenous lipid is measured. The protocol has been validated for the use of human monocytes purified from individuals with HIV infection and elderly HIV uninfected individuals. This model is versatile and allows monocyte transmigration and foam cell formation to be evaluated using either microscopy or flow cytometry as well as allowing the assessment of atherogenic factors present in serum or plasma.

We describe a protocol to measure transmigration by monocytes across human endothelial monolayers and their subsequent maturation into foam cells. This provides a versatile method to assess the atherogenic properties of monocytes isolated from people with different disease conditions and to evaluate factors in blood which may enhance this propensity.

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Coronary artery disease (CAD) is a leading cause of morbidity and mortality worldwide. Atherosclerosis, a leading cause of CAD, is initiated by the ...

Characterization of Human Monocyte Subsets by Whole Blood Flow Cytometry Analysis

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can have pathological significance. An ideal method for examining alterations to monocytes is whole blood flow cytometry as the minimal handling of samples by this method limits artifactual cell activation. However, many different approaches are taken to gate the monocyte subsets leading to inconsistent identification of the subsets between studies. Here we demonstrate a method using whole blood flow cytometry to identify and characterize human monocyte subsets (classical, intermediate, and non-classical). We outline how to prepare the blood samples for flow cytometry, gate the subsets (ensure contaminating cells have been removed), and determine monocyte subset expression of surface markers &#8212; in this example M1 and M2 markers. This protocol can be extended to other studies that require a standard gating method for assessing monocyte subset proportions and monocyte subset expression of other functional markers.

Here we present a protocol for characterizing monocyte subsets by whole blood flow cytometry. This includes outlining how to gate the subsets and assess their expression of surface markers and giving an example of the assessment of the expression of M1 (inflammatory) and M2 markers (anti-inflammatory).

全血流式细胞术分析人单核细胞亚群的表征

Monocytes are key contributors in various inflammatory disorders and alterations to these cells, including their subset proportions and functions, can ...

Isolation of Monocytes from Whole Blood by Immunomagnetic Negative Selection

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