Name: neutrophil isolation protocol
Uploaded: 2008-07-23T22:05:00
Description: Watch this Scientific Journal Video about Neutrophil Isolation Protocol at JoVE.com

<p class="jove_content">Funding from NIH R01 HL56621.</p>

<p class="jove_title">&nbsp;Neutrophil Isolation Protocol</p>
<ol>
<li>Bring all reagents to room temperature. </li>
<li>Collect 5.0 ml of neutrophil isolation media in centrifuge tube. Carefully layer 5.0 ml of blood over the separation media. Perform this step slowly and carefully, and with the pipette tip close to the surface of the media to avoid mixing the blood and the media.</li>
<li>Centrifuge at 500 RCF for 35 min at 20-25&deg;C. The blood should separate out into 6 distinct bands: plasma, monocytes, isolation media, neutrophils, more ...

<ol>
	<li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+leucocytes+from+blood+and+bone+marrow.">Separation of leucocytes from blood and bone marrow.</a> <em style='font-style: italic'>Scand. J. Clin. Invest</em>. <b>21</b>, 77-83 (1968).</li><li>England, J. M., Rowan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assignment+of+values+to+fresh+blood+used+for+calibrating+automated+blood+cell+counters.">The assignment of values to fresh blood used for calibrating automated blood cell counters.</a> <em style='font-style: italic'>Clin. Lab. Haemat</em>. <b>10</b>, 203-212 (1988).</li><li>Garcia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+leukocyte+extraction+methods+for+cytomegalovirus+antigenemia+assay.">Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay.</a> <em style='font-style: italic'>J. Clin. Microbiol</em>. <b>34</b>, 182-184 (1996).</li><li>Ferrante, A., Thong, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+conditions+for+simultaneous+purification+of+mononuclear+and+polymorphonuclear+leucocytes+from+human+peripheral+blood+by+the+Ficoll-Hypaque+method.">Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method.</a> <em style='font-style: italic'>J. Immunol. Methods</em>. <b>36</b>, 109-109 (1980).</li></ol>

<p class="jove_content">The density gradient separation method is used to isolate human neutrophils from whole blood using a mixture of sodium metrizoate and Dextran 500. This method is based on the mononuclear leukocyte separation method by Boyum (1968) which was modified for neutrophil separation by Ferrante and Thong (1980). </p><p class="jove_content">
After collection from a donor, whole blood may be anticoagulated with EDTA, citrate, or heparin. Since they are short-lived, neutrophils should be used within 2-4 hours of collection. The pro...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Lymphocyte Poly(R)</td>
<td>Reagent</td>
<td>Cedarlane Labs</td>
<td>&nbsp;</td>
<td>PolymorphprepTM can be used as an alternative</td>
</tr>
<tr>
<td>Hank's Balanced Salt Solution without Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Human Serum Albumin</td>
<td>Reagent</td>
<td>ZLB Bioplasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Red Cell Lysis Buffer</td>
<td>Reagent</td>
<td>Roche Diagnostics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortexer</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Pipettes and bulb</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PolymorphprepTM</td>
<td>Reagent</td>
<td>Axis-Shield PoC</td>
<td>&nbsp;</td>
<td>Alternative reagent to Lymphocyte-Poly (R)</td>
</tr>
<tr>
<td>Syringe Filter</td>
<td>Other</td>
<td>Millipore</td>
<td>SLGV033RS</td>
<td>Millex-GV, 0.22 &#181;m, PVDF, 33 mm, gamma-sterilizable</td>
</tr>
<tr>
<td>Hank's Balance Salt Solution with Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
	

neutrophil isolation protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Watch this Scientific Journal Video about Neutrophil Isolation Protocol at JoVE.com

Neutrophil Isolation Protocol

<p class="jove_content">Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.</p>

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Research

JoVE Journal

Biology

Protocol for Mosquito Rearing (A. gambiae)

Protocol for Mosquito Rearing A. gambiae

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Protokol til Isolering af primære humane hepatocytter og Tilsvarende Major populationer af ikke-parenchymale leverceller

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

Clock Scan Protocol for Image Analysis: ImageJ plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

En simpel højeffektiv protokol til den pankreatiske ø-isolation fra mus

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

The MUB<sub>40</sub> Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

MUB<sub>40</sub> peptid til brug i forbindelse med afsløring neutrofile-medieret betændelse begivenheder

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Hurtig Lipid Droplet Isolation protokollen ved hjælp af en almindelig Organelle isolering Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

En forbedret tids- og arbejdseffektiv protokol til musens primære hepatocytisolering

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Neutrophil Lifespan Extension with CLON-G and an <em>In Vitro</em> Spontaneous Death Assay

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

Neutrofil levetidsforlængelse med CLON-G og en <em>in vitro</em> spontan dødsanalyse

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...