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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5 minutes

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 needle and replace it with the filter.Inject HSA through filter into HBSS vial vortex two mix when you have your other materials ready and at room temperature, acquire about 15 milliliters of whole blood. This will yield 10 to 15 milliliters of 10 to the eighth neutrophils. The donor should not have consumed alcohol or over the counter drugs for 24 hours prior to donating the blood sample.These can disrupt the separation process. Whole blood may be anticoagulated with EDTA citrate or heparin by adding nine parts blood to one part K three EDTA sodium citrate, or heparin following manufacturer's instructions. The following steps will be described in this procedure.Separate and remove plasma and monocytes. Separate and acquire a neutrophil layer lysing red blood cells free suspend neutrophils first separation. Acquire neutrophils in NIM layer.Collect five milliliters of isolation media in centrifuge tube. Placing the isolation media lower in the tube allows it to act as a filter, as the centrifuge will force the blood down through it Carefully. Layer five milliliters of blood over the NIM for clean separation.Be very careful to avoid mixing the solutions. Perform this step slowly and carefully and with your pipette tip close to the surface of the resolving media to prevent mixing centrifuge. The solution at 500 RCF for 35 minutes.At 20 to 25 degrees Celsius, the blood should separate out into six distinct bands. The six bands are plasma monocytes, isolation media, neutrophils, more isolation media, and the red blood cell pellet at the bottom. If these six bands are not clear and distinct, the separation process was not clean and would need to be repeated.Common causes of bad separation include old isolation media donor having consumed alcohol in the past 72 hours or donor having consumed other medicine such as Tylenol or aspirin. Carefully remove and discard the plasma monocytes and isolation media. Place these layers in a sealable tube and discard carefully pipette the layer of neutrophils and all of the isolation media beneath the neutrophils into a clean centrifuge file to recover as many neutrophils as possible.It's often easiest to include some of the isolation media from the layer below as well. Do not disturb the pallet at the bottom. Second declaration, refine neutrophil layer.Dilute the neutrophil solution to 10 milliliters with HBSS without calcium. Invert the tube a few times to suspend the cells centrifuge. The neutrophil solution.350 RCF for 10 minutes. At 20 to 25 degrees Celsius, a red pellet should be present on the bottom of the tube containing neutrophils and residual red blood cells. Remove the supra natin with the pipette.Be careful. to disturb the pellet at the bottom. Lysing the red blood cells.The red blood cells in the sample must now be destroyed by lysis. To lys the residual red blood cells, add two milliliters of red cell lysis buffer to the tube to resus. Suspend the pellet vortex the vial at a setting of three to four.Avoid increasing the vortex setting above four 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 palate. Centrifuge the lysis solution at 250 RCF for five minutes.Use the soft start option to minimize damage to the sample. Remove the supra natin with pipette. Repeat the lysing process.If require, release suspension of neutrophils, add 500 microliters HBSS without calcium to each tube. Vortex the pellet at a setting of three to four, dilute to 10 milliliters with HPSS without calcium centrifuge. The neutrophils at 250 RCF for five minutes aspirate the snat and discard.Re suspend the pellet in 250 microliters of HBSS with HSA solution, two times 10 to the six. Neutrophils per milliliter are typically collected.

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JoVE Journal

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Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

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We describe here a method for obtaining and identifying a newly characterized subpopulation of neutrophil-derived giant phagocytes. These cells develop in culture from fresh human blood neutrophils, and are characterized by phagocytosis, autophagy, immensely large size, and extended lifespan. This method is essential to further investigate this unique neutrophil-derived subpopulation.

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This protocol details a method for the isolation of neutrophils from whole blood, buffy coats, or leukapheresis membranes, achieving good yield, high purity, and minimal cell activation. We utilize gradient purification, red blood cell (RBC) sedimentation, and RBC lysis to obtain a high-quality/purity neutrophil preparation.

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This defined protocol describes a real-time high-throughput microscopy approach to visualize and quantify human neutrophil extracellular trap (NET) release in vitro. The reproducible method allows investigation of the characteristics and kinetics of NET release upon stimulation with distinct NETosis inducers and enables assessment of the pharmacology of NETosis antagonists.

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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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