Name: Neutrophil Isolation Protocol (Video) | JoVE Journal
Uploaded: 2008-07-23T22:05:00.000+00:00
Duration: 7 min 12 s
Description: Learn a reliable density gradient method to isolate human neutrophils from whole blood for immunology research. This standardized protocol enables reproducible study of inflammatory immune responses.

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Funding from NIH R01 HL56621.

&nbsp;Neutrophil Isolation Protocol
<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 isolation media, and the red blood cell pellet (Figure 1). If these bands are not clear, the separation process was not clean and will need to be repeated.</li>
<li>Carefully remove the top three layers (plasma, monocytes, and isolation media) using a pipette. Dispose of these layers.</li>
<li>Carefully pipette the layer of neutrophils and all of the isolation media beneath the neutrophils. Place the solution into a clean centrifuge tube.</li>
<li>Dilute the neutrophil solution to 10 ml with HBSS without Ca2+/Mg2+. Invert the tube a few times to suspend the cells. </li>
<li>Centrifuge the neutrophil solution at 350 RCF for 10 minutes. A red pellet should be present at the bottom of the tube, containing neutrophils and residual red blood cells (RBCs). Remove the supernatant with a pipette carefully so that the pellet is not disturbed.</li>
<li>To lyse the residual RBCs, add 2 ml Red Cell Lysis Buffer to the tube. To resuspend the pellet, vortex the vial at a setting of 3-4. Avoid increasing the vortex setting above 4, since this may cause the neutrophils to activate. It may be necessary to vortex for several seconds, or to "pulse" the vortex to dissolve the pellet. </li>
<li>Centrifuge the tube at 250 RCF for 5 min. Remove the supernatant with pipette. Repeat the lysing process if required.</li>
<li>Add 500 &mu;l HBSS without Ca2+/Mg2+ to each tube. Again, vortex to resuspend the pellet at a setting of 3-4. Dilute to 10 ml with HBSS without Ca2+/Mg2+.</li>
<li>Centrifuge the tubes at 250 RCF for 5 min. Aspirate the supernatant and discard.</li>
<li>Resuspend the pellet in 250 &mu;l HBSS/HSA Solution (2% HSA). Cells may then be counted and adjusted to desired concentration.</li>
</ol>
&nbsp;

<ol><li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Separation+of+leucocytes+from+blood+and+bone+marrow%5BTitle%5D)+AND+%22Scand.+J.+Clin.+Invest%22%5BJournal%5D)">Separation of leucocytes from blood and bone marrow.</a> Scand. J. Clin. Invest. 21, Suppl 97. 77-83 (1968).</li>
<li>England, J. M., Rowan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+assignment+of+values+to+fresh+blood+used+for+calibrating+automated+blood+cell+counters%5BTitle%5D)+AND+%22Clin.+Lab.+Haemat%22%5BJournal%5D)">The assignment of values to fresh blood used for calibrating automated blood cell counters.</a> Clin. Lab. Haemat. 10, 203-212 (1988).</li>
<li>Garcia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+two+leukocyte+extraction+methods+for+cytomegalovirus+antigenemia+assay%5BTitle%5D)+AND+%22J.+Clin.+Microbiol%22%5BJournal%5D)">Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay.</a> J. Clin. Microbiol. 34, 182-184 (1996).</li>
<li>Ferrante, A., Thong, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimal+conditions+for+simultaneous+purification+of+mononuclear+and+polymorphonuclear+leucocytes+from+human+peripheral+blood+by+the+Ficoll-Hypaque+method%5BTitle%5D)+AND+%22J.+Immunol.+Methods%22%5BJournal%5D)">Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method.</a> J. Immunol. Methods. 36, 109-109 (1980).</li>
</ol>

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). 
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...

<table><tbody><tr><td>Lymphocyte Poly(R)</td><td>Reagent</td><td>Cedarlane Labs</td><td></td><td>PolymorphprepTM can be used as an alternative</td></tr><tr><td>Hank&amp;rsquo;s Balanced Salt Solution without Calcium Chloride</td><td>Reagent</td><td>Invitrogen</td><td></td><td></td></tr><tr><td>Human Serum Albumin</td><td>Reagent</td><td>ZLB Bioplasma</td><td></td><td></td></tr><tr><td>Red Cell Lysis Buffer</td><td>Reagent</td><td>Roche Group</td><td></td><td></td></tr><tr><td>Centrifuge</td><td>Tool</td><td></td><td></td><td></td></tr><tr><td>Vortexer</td><td>Tool</td><td></td><td></td><td></td></tr><tr><td>Pasteur Pipettes and bulb</td><td>Tool</td><td></td><td></td><td></td></tr><tr><td>PolymorphprepTM</td><td>Reagent</td><td>Axis-Shield</td><td></td><td>Alternative reagent to Lymphocyte-Poly (R)</td></tr><tr><td>Syringe Filter</td><td>Other</td><td>EMD Millipore</td><td>SLGV033RS</td><td>Millex-GV, 0.22 &amp;#x03BC;m, PVDF, 33 mm, gamma-sterilizable</td></tr><tr><td>Hank&amp;rsquo;s Balance Salt Solution with Calcium Chloride</td><td>Reagent</td><td>Invitrogen</td><td></td><td></td></tr></tbody></table>

neutrophil isolation protocol

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.

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.

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

Neutrophil isolation protocol. This procedure will extract white blood cells from a whole blood sample equipment centrifuge and vials. Vortex sir needle and syringe syringe filter.Milli pour brand MX gv 0.22 Micrometer materials. Hank spell and salt solution neutrophil separation requires two kinds of HBSS solutions. HBSS with calcium chloride will be used to make a solution with human serum albumin.HBSS without calcium chloride will be used to suspend and wash the neutrophils. Please take special care to use the appropriate HBSS solution in this procedure. Human serum albumin or HSA is a protein in the blood plasma that maintains pH and SMO pressure.Do not use bovine serum amalgamate red cell lysis buffer. This is manufactured by Roche Diagnostics, neutrophil Isolation Media NIM Cedar Line Labs Lymph light Poly separation media protected from light and kept in refrigerator for best results. All reagents should be at room temperature at time of use.Remove reagents from refrigerator one hour before experiment. Prepare H-B-S-S-H-S-A solution for neutrophil separation. Pipette HBSS with calcium chloride into vial.Draw HSA into syringe. Avoid trapping air bubbles. Remove the syringe ne

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Learn a reliable density gradient method to isolate human neutrophils from whole blood for immunology research. This standardized protocol enables reproducible study of inflammatory immune responses.

Neutrophil Isolation Protocol

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