Name: Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils (Video) | JoVE Journal
Uploaded: 2025-11-11T14:30:00.000+00:00
Duration: 8 min 14 s
Description: Learn to isolate pure low-density neutrophils using density-gradient centrifugation and magnetic microbead separation. This efficient method enables functional studies of LDN in immunology and disease research.

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5 minutes

The authors thank C&#233;sar D&#237;az-God&#237;nez (Instituto de Investigaciones Biom&#233;dicas-UNAM) for his help with fluorescent microscopy to visualize NETs. This research was supported by grant PAPIIT IN205523 from Direcci&#243;n General de Asuntos del Personal Acad&#233;mico, Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM), Mexico, and by grant CF-2023-I-610 from Secretar&#237;a de Ciencias, Humanidades, Tecnolog&#237;a e Inovaci&#243;n (Secihti), Mexico.

All procedures in this protocol follow the guidelines of the Human Research Bioethics Committee at Instituto de Investigaciones Biom&#233;dicas - Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM). All participants provided informed consent.
1. Isolation of peripheral blood mononuclear cells (PBMC) and neutrophils from human blood
<ol>
	<li>Obtain about 10 mL of blood from a healthy adult volunteer by venipuncture. Add 10 U/mL of heparin as an anticoagulant. 
	&#8203;CAUTION: Dispose of the needle into a biohazard needle disposal container. Syringes and other materials in touch with blood should be placed in a bag and autoclaved before disposal.</li>
	<li>In a 15 mL conical centrifuge tube, add 2 mL of 6% dextran T500 in PBS. Then add 10 mL of blood by draining it down the side of the tube. Invert the tube a couple of times to mix the blood and dextran.</li>
	<li>Let the tube rest for 45 min, while erythrocytes sediment. The leukocyte-rich plasma appears above the erythrocyte layer.</li>
	<li>In another 15 mL centrifuge tube, add 5 mL of density gradient medium. Carefully pipette the plasma, without touching the erythrocytes, and layer it on top of the medium. Two separate phases must be formed.</li>
	<li>Centrifuge the tube at 516 x g for 20 min at 4 &#176;C. Mononuclear cells (PBMC) form a band between plasma and the medium layers. Neutrophils form a pellet at the bottom (Figure 1).</li>
	<li>Isolation of PBMC
	<ol>
		<li>Eliminate by aspiration the plasma on top of the PBMC without touching the cells. Collect the cells from the band between the plasma and the medium. Make sure to aspirate as little medium as possible.</li>
		<li>Place cells into a 50 mL conical centrifuge tube. Add 20 mL of PBS. Centrifuge the tube at 400 x g for 5 min at 4 &#176;C.</li>
		<li>Carefully aspirate the supernatant and scrape the tube to separate the cells. Add 10 mL of cold PBS to resuspend the cells. Count the PBMC using a Neubauer chamber. 
		NOTE: Do not use a pipette to resuspend the cells, because this may damage the cells.</li>
	</ol>
	</li>
	<li>Isolation of neutrophils
	<ol>
		<li>Remove the density gradient medium. Separate the cells by scraping the tube and add 10 mL of cold PBS.</li>
		<li>Put the cells into a fresh 50 mL centrifuge tube and centrifuge at 400 x g for 5 min at 4 &#176;C. Aspirate the supernatant and separate the cells as described in step 1.6.3.</li>
	</ol>
	</li>
	<li>To eliminate residual erythrocytes, add 10 mL of cold hypotonic solution (0.2% NaCl, 1% BSA, 20 mM Hepes, pH = 7.4), and mix gently for exactly 1 min.</li>
	<li>Quickly add 10 mL of cold hypertonic solution (1.6% NaCl, 1% BSA, 20 mM HEPES, pH = 7.4) to make the solution isotonic.</li>
	<li>Count the neutrophils using a Neubauer chamber (purity &#62; 95%). Pellet the cells by centrifuging as in step 1.6.2. and resuspend them in cold PBS. Maintain the tube on ice. 
	NOTE: It is reported that the half-life of neutrophils in vitro is 8 - 12 h46,47,48. In our experience with this protocol, neutrophils maintain their functions for approximately 6 to 8 h.</li>
</ol>
2. Purification of low-density neutrophils (LDN)
<ol>
	<li>Centrifuge PBMC at 400 x g for 5 min at 4 &#176;C. Remove supernatant and resuspend cells in 120 &#181;L of cold wash buffer (1% BSA in PBS).</li>
	<li>Add 35 &#181;L of CD66b magnetic microbeads. Incubate the mixture in the dark at 4 &#176;C for 30 min. Mix gently every 10 min to prevent cell aggregation.</li>
	<li>Add 1 mL of cold wash buffer. Place cells in a microcentrifuge and spin at 400 x g for 3 min.</li>
	<li>Remove supernatant, separate the cell pellet by scraping the tube, and resuspend cells in 1 mL of wash buffer.</li>
	<li>Place a magnetic separation column onto a magnet. Add 0.5 mL of wash buffer to the column and let it pass through the column.</li>
	<li>Transfer cells (1 mL) onto the column. Let the buffer pass through the column drop by drop.</li>
	<li>Add 0.5 mL of wash buffer into the column and let it pass through. Add another 0.5 mL of wash buffer into the column.</li>
	<li>Remove the column from the magnet and put it in a microcentrifuge tube. Add 1 mL of wash buffer to the column.</li>
	<li>Insert the plunger on top of the column and gently apply pressure to elute the cells. Remove the plunger and put the column on top of a new microcentrifuge tube.</li>
	<li>Add another 1 mL of wash buffer. Insert the plunger on top of the column and gently apply pressure to elute the cells.</li>
	<li>Place both tubes in a microcentrifuge and spin them at 800 x g for 3 min. Finally, resuspend the cells (LDN) from both tubes into 1 mL of cold PBS. Keep on ice. 
	NOTE: The first 2 mL of flow-through contains the rest of the PBMC. These cells can be used for other studies.</li>
</ol>
3. Multicolor flow cytometry
<ol>
	<li>Resuspend purified cells at 1 x 106 cell/mL in labeling buffer (1% FBS in PBS). Add 250 &#181;L of cells into a 1.5 mL microcentrifuge tube.</li>
	<li>Add the corresponding antibodies against neutrophil membrane molecules (for details, see Table of Materials). Incubate the cells for 30 min at 4 &#176;C, protecting them from light.</li>
	<li>Add 1 mL of PBS. Place tubes in a microcentrifuge and spin them at 800 x g for 3 min.</li>
	<li>Aspirate the supernatant, break the cell pellet by tapping the tube, and resuspend cells in 0.5 mL of 1% paraformaldehyde.</li>
	<li>Keep the cells at 4 &#176;C protected from light, until analyzed by flow cytometry. Analyze cells by flow cytometry, capturing 10,000 events per sample. Perform cell sorting as previously described22. 
	CAUTION: Paraformaldehyde is an irritant to the skin and the respiratory tract. Make sure you do not breathe the vapors.</li>
</ol>
4. Detection of reactive oxygen species (ROS)
<ol>
	<li>In a 1.5 mL microcentrifuge tube, add 2.5 x 105 cells. Place tubes in a microcentrifuge and spin at 800 x g for 3 min.</li>
	<li>Aspirate the supernatant, scrape the tube to separate the cells, and resuspend the cells in 100 &#181;L of 15 &#181;M dihydrorhodamine 123 in PBS.</li>
	<li>Incubate the cells protected from light at 37 &#176;C for 20 min. Add 1 mL of PBS.</li>
	<li>Place tubes in a microcentrifuge and spin at 800 x g for 3 min. Aspirate the supernatant, scrape the tube to separate the cells, and resuspend them in 100 &#181;L of 50 nM phorbol 12-myristate 13-acetate (PMA) in PBS.</li>
	<li>Incubate the cells protected from light at 37 &#176;C for 45 min. Add 1 mL of PBS.</li>
	<li>Place tubes in a microcentrifuge and spin them at 800 x g for 3 min. Aspirate the supernatant, scrape the tube to separate the cells, and resuspend them in 0.5 mL of 1% paraformaldehyde in PBS.</li>
	<li>Keep the cells at 4 &#176;C protected from light until analyzed by flow cytometry. Analyze cells by flow cytometry, capturing 10,000 events per sample.</li>
</ol>
5. Visualization of neutrophil extracellular traps (NETs)
<ol>
	<li>Place coverslips in wells of a 12-well plate and cover them with 10 &#181;g/mL poly-L-Lysine. Incubate the plate overnight at 4 &#176;C with gentle agitation.</li>
	<li>Remove the poly-L-Lysine, and wash the coverslips with PBS, incubating the plate for 5 min at room temperature with gentle agitation. Wash the coverslips with PBS two more times.</li>
	<li>Remove the PBS and let the coverslips dry by placing the plate inside a laminar-flow hood for 2 h.</li>
	<li>Resuspend LDN at 1 x 106 cell/mL in RPMI-1640 medium with 5% FBS. Take 350 &#181;L (3.5 x 105 cells) of the cell suspension and put them into a well of the 12-well plate containing the coverslips.</li>
	<li>Keep the plate for 30 min in a 5% CO2 incubator at 37 &#176;C. Add 3.5 &#181;L of 5 &#181;M PMA diluted in PBS to each well (final concentration of PMA is 50 nM).</li>
	<li>Keep the plate for 4 h in a 5% CO2 incubator at 37 &#176;C. Add 350 &#181;L of 8% paraformaldehyde in PBS, and keep the plate overnight in a 5% CO2 incubator at 37 &#176;C.</li>
	<li>Place a transparent film over a test tube stand as previously described49, so that wells are formed on the tube holes.</li>
	<li>Fill each well with the various solutions to wash or stain the coverslips, forming a big drop. Remove the coverslips and put them upside down on a drop of water for 5 min. In the same manner, wash the coverslips three more times with water.</li>
	<li>Place the coverslips upside down on a drop of blocking buffer (5% BSA in PBS) and incubate for 20 min at room temperature.</li>
	<li>Transfer the coverslips to a drop of the corresponding primary antibody (e.g., anti-elastase or anti-citrulline) in blocking buffer and incubate for 60 min at room temperature.</li>
	<li>Wash coverslips 2x in 0.01% Tween-20 in PBS. Transfer the coverslips to a drop of the corresponding secondary antibody in blocking buffer containing 150 nM DAPI, and incubate for 60 min at room temperature, protected from light.</li>
	<li>Wash coverslips 2x in 0.01% Tween-20 in PBS. Place a drop of antifade mounting medium onto a glass slide and put coverslips upside down.</li>
	<li>Seal coverslips around the perimeter with nail polish. Store mounted slides at 4 &#176;C, protected from light. Visualize NETs using a fluorescence microscope.</li>
</ol>
6. Detection of neutrophil extracellular traps (NETs)
<ol>
	<li>Resuspend cells at 5 x 105 cell/mL in 500 nM Sytox Green, diluted in RPMI-1640 medium with 5% FBS.</li>
	<li>Take 100 &#181;L (5 x 104 cells) of the cell suspension and put them into a well of a 96-well tissue culture plate.</li>
	<li>Keep the plate for 20 min in a 5% CO2 incubator at 37 &#176;C. Add to each well 20 &#181;L of 300 nM PMA diluted&#160;in RPMI-1640 medium (final concentration of PMA is 50 nM).</li>
	<li>Transfer the plate to a pre-warmed microplate reader. Incubate the plate at 35 &#176;C for 4 h, taking fluorescence measurements from the bottom of the plate every 5 min (using the 485 nm excitation and 528 nm emission filters).</li>
</ol>

The authors state that this study was carried out without any commercial or financial ties that might be perceived as a potential conflict of interest.

Generally, neutrophils were thought of as homogeneous cells. However, recent evidence has shown that neutrophils could exist as cells with different activation states and/or multiple phenotypes. Thus, various neutrophil subpopulations may exist13,14,15. One neutrophil subpopulation, the low-density neutrophils (LDN), has acquired great interest due to their particular intrinsic properties and also because they increase in multip...

Neutrophils, the predominant leukocytes in human blood1, are key participants of the innate immune system. They arrive in large numbers to tissues with inflammation or infection2. There, neutrophils activate several effector functions, such as phagocytosis3,4, degranulation5,6, and formation of neutrophil extracellular traps (NETs)7. Additionally, neutrophils also participate in the adaptive immune response8. The classical view of neutrophils considers them as homogeneous cells produced in the bone marrow with predetermined responses9,10. However, recent studies reveal that neutrophils are heterogeneous cells with multiple phenotypic and functional states under both healthy and disease states11,12,13,14,15.
Among several neutrophil subpopulations, the low-density neutrophils (LDN) have attracted much interest because of their intrinsic properties and because they increase in numbers in several diseases16,17,18. LDN were found in the blood of systemic lupus erythematosus (SLE) patients in 198619. They were detected following the process for separating leukocytes in a density gradient20,21. After centrifugation of blood or leukocyte-rich plasma on a density medium (e.g., Ficoll-Paque), monocytes and lymphocytes (known as peripheral blood mononuclear cells [PBMC]) form a band in the upper (low-density) part of the tube. Neutrophils sediment at the bottom of the tube. Among the PBMC, a few neutrophils were also found; these are LDN (Figure 1). Small numbers of LDN are in the blood of healthy individuals22. However, LDN numbers increase significantly in multiple immunosuppression and chronic inflammation conditions, including SLE23,24, sepsis25, psoriasis26, asthma27, juvenile idiopathic arthritis28, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis29, HIV infection30, malaria31 and tuberculosis32. Although LDN numbers increase in all the mentioned pathologies, LDN&#160;have been mostly studied in the context of SLE17. LDN from the blood of SLE patients seems to have a greater capacity to form NETs33, to secrete large amounts of proinflammatory mediators34, and to activate T cells24. However, in other conditions, such as cancer, LDN&#160;are reported to consist of mature and immature neutrophils35, with T-cell suppressive properties36,37,38. Similarly, in COVID-19 patients, LDN&#160;are also reported to suppress T cell proliferation39, but contrary to SLE, LDN seems to produce fewer NETs39. Therefore, the origin, composition, and functional properties of LDN are still controversial.
Because LDN are found together with PBMC, studying them is technically complicated. To fully explore LDN properties in different pathologies, it is necessary to purify them. A common procedure of LDN separation is fluorescent cell sorting22,40,41,42,43,44,45. However, cell sorting requires a long time for cell separation. This may lead to cell loss or low recovery if the sorting time is not sufficient. In addition, cells are subjected to excessive stress, causing variable results while studying the function of these cells. Long sorting time also increases the cost of experimental procedures and requires specialized personnel.
Here, we present a rapid and efficient method for obtaining large numbers of pure and viable human LDN in a very short time. After density-gradient centrifugation, LDN&#160;are separated by magnetic cell sorting (MACS; Figure 1). The main advantage of this purification method is that LDN&#160;are separated with high purity (more than 90%) and viability (more than 96%). Also, purified LDN are&#160;completely functional as indicated by their&#160;capacity to generate reactive oxygen species (ROS) and to release NETs. This protocol can be easily implemented in any laboratory interested in neutrophil biology to quickly separate LDN from the blood of people with multiple diseases in order to further study these cells.

<table><tbody><tr><td>&amp;nbsp;Anti-Citrulline rabbit polyclonal antibody</td><td>AbCam</td><td>ab100932</td><td>1/50 dilution</td></tr><tr><td>96-well tissue culture plate</td><td>Costar; Corning, Inc.</td><td>3596</td><td></td></tr><tr><td>Alexa Fluor 488 anti-human CD11b mouse IgG1 antibody</td><td>BioLegend</td><td>301317</td><td>0.25 &amp;mu;g/mL</td></tr><tr><td>Alexa Fluor 647 anti-human CD66b mouse IgM antibody</td><td>BioLegend</td><td>305109</td><td>0.05 &amp;mu;g/mL</td></tr><tr><td>Anti-Mouse IgG (H+L) goat antibody, Alexa Fluor 488</td><td>Invitrogen - Thermo Fisher Scientific</td><td>A-11001</td><td>1/50 dilution</td></tr><tr><td>Anti-Neutrophil Elastase mouse monoclonal IgG1 antibody</td><td>Santa Cruz Biotechnology</td><td>sc-55549</td><td>10 &amp;micro;g/mL</td></tr><tr><td>Anti-Rabbit IgG (H+L) donkey antibody, Alexa Fluor 555</td><td>Invitrogen - Thermo Fisher Scientific</td><td>A-31572</td><td>1/50 dilution</td></tr><tr><td>APC anti-human CD62L mouse IgG1 antibody</td><td>BioLegend</td><td>304809</td><td>0.03 &amp;mu;g/mL</td></tr><tr><td>APC/Cyanine7 anti-human CD14 mouse IgG1 antibody</td><td>BioLegend</td><td>367107</td><td>0.25 &amp;mu;g/mL</td></tr><tr><td>Attune NxT flow cytometer&amp;nbsp;</td><td>Thermo Fisher Scientific, Inc.</td><td>(blue/red lasers)</td><td></td></tr><tr><td>Bovine serum albumin (BSA) Fraction V</td><td>MP Biomedicals</td><td>810025</td><td></td></tr><tr><td>Brilliant Violet 510 anti-human CD33 mouse IgG1 antibody</td><td>BioLegend</td><td>366609</td><td>0.30 &amp;mu;g/mL</td></tr><tr><td>DAPI</td><td>Calbiochem/EMD Millipore</td><td>268298</td><td></td></tr><tr><td>Dextran T500</td><td>Pharmacosmos A/C</td><td>5.51005E+11</td><td></td></tr><tr><td>Dihydrorhodamine-123</td><td>Anaspec, Inc.</td><td>AS-85711</td><td></td></tr><tr><td>FACS cell sorter</td><td>Becton Dickinson&amp;nbsp;</td><td>Model FACSAria</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco</td><td>16000-044</td><td></td></tr><tr><td>Ficoll-Paque</td><td>Merck KG</td><td>GE 17-1440-03</td><td></td></tr><tr><td>FITC anti-human CD98 mouse IgG1 antibody</td><td>BioLegend</td><td>315603</td><td>0.30 &amp;mu;g/mL</td></tr><tr><td>FlowJo Software</td><td>Becton Dickinson</td><td>Version 10, 2019</td><td></td></tr><tr><td>Fluorescence inverted microscope</td><td>Olympus</td><td>Model IX-70</td><td></td></tr><tr><td>Heparin</td><td>Inhepar - PiSA Farmaceutica</td><td>3660149</td><td>5000 UI/mL</td></tr><tr><td>Hepes</td><td>Sigma Aldrich</td><td>H7006</td><td></td></tr><tr><td>MACSprep Chimerism CD66b Microbeads</td><td>Miltenyi Biotec</td><td>130-111-552</td><td></td></tr><tr><td>Magnet</td><td>Miltenyi Biotec</td><td>130-042-102</td><td></td></tr><tr><td>Magnetic separation column</td><td>Miltenyi Biotec</td><td>130-042-201</td><td></td></tr><tr><td>Microcentrifuge</td><td>Eppendorf</td><td>Model 5415C</td><td></td></tr><tr><td>Microplate reader</td><td>BioTek Instruments</td><td>Model Synergy HT</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma Aldrich</td><td>158127</td><td></td></tr><tr><td>PE anti-human CD10 mouse IgG1 antibody</td><td>BioLegend</td><td>312203</td><td>0.05 &amp;mu;g/mL</td></tr><tr><td>PE anti-human CD16b mouse IgG2a antibody</td><td>BD Pharmingen</td><td>550868</td><td>1/40 dilution</td></tr><tr><td>PE/Cyanine5 anti-human CD15 mouse IgG1 antibody</td><td>BioLegend</td><td>323013</td><td>0.25 &amp;mu;g/mL</td></tr><tr><td>Phorbol 12-myristate 13-acetate (PMA)</td><td>Sigma Aldrich</td><td>P8139</td><td></td></tr><tr><td>poly-L-Lysine</td><td>Sigma Aldrich</td><td>P2658</td><td></td></tr><tr><td>Refrigerated centrifuge</td><td>Eppendorf</td><td>Model 5702R</td><td></td></tr><tr><td>RPMI-1640 tissue culture medium</td><td>Gibco</td><td>23400-021</td><td></td></tr><tr><td>SYTOX Green</td><td>Molecular Probes, Inc.</td><td>S-7020</td><td></td></tr><tr><td>Tween-20&amp;nbsp;</td><td>Sigma Aldrich</td><td>P2287</td><td></td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td></td></tr></tbody></table>

rapid magnetic-microbead method for efficient purification of low-density neutrophils

Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils

Neutrophils, important leukocytes of innate immunity, have traditionally been considered a homogeneous cell population. Nevertheless, recent evidence has shown that neutrophils exist in several subpopulations. One such subpopulation is low-density neutrophils (LDN). LDN are&#160;found in small numbers in the blood of healthy individuals, but their&#160;numbers increase significantly in diseases such as systemic lupus erythematosus, autoimmune disorders, cancer, and infections. In these cases, LDN may participate in the pathogenesis of the disease. The only way to isolate LDN is through density-gradient centrifugation of peripheral blood. However, after centrifugation, LDN co-purify&#160;with mononuclear cells. Thus, studying this neutrophil subpopulation is challenging. There is no standard methodology to separate LDN from mononuclear cells. Typically, LDN&#160;are separated by cell sorting in a flow cytometer. However, this method requires long sorting times (hours) to obtain enough pure cells for further functional studies. This seriously affects the viability and function of cells. Here, we propose a practical method to obtain large numbers of pure and viable LDN in a short time. After density-gradient centrifugation, the mononuclear cell fraction is incubated with anti-CD66b magnetic microbeads, and then LDN are separated through magnetic columns in &#60; 30 min. Purified LDN (CD66b+ cells) are labelled with monoclonal antibodies against CD10, CD11b, CD14, CD15, CD16b, CD33, CD62L, CD66b, and CD98, and are analyzed by flow cytometry for confirmation. Purified LDN&#160;are completely functional as indicated by their capacity to produce reactive oxygen species, and to form neutrophil extracellular traps. This new purification method results in LDN with high purity (more than 90%) and viability (more than 96%) in a short time period. This method can easily be scaled up to obtain large numbers of pure LDN to evaluate LDN functions in different diseases through biochemical or other omics analysis.

Low-density neutrophils (LDN) in healthy individuals represent around 5% of the PBMC (Figure 2A). The protocol described here typically delivers pure (&gt; 90%) LDN. Cells are also efficiently recovered (yield around 98%) with high viability (&gt; 95%; Figure 2B). For comparison, LDN were also purified by fluorescent-activated cell sorting as previously described22. Briefly, PBMC were labelled with anti-CD...

Low-density neutrophils (LDN) increase significantly in several diseases. LDN are usually isolated through cell sorting. We present a practical method to obtain pure and viable LDN. After density-gradient centrifugation of peripheral blood, cells are incubated with magnetic microbeads, and then LDN are separated through magnetic columns.

Neutrophils, important leukocytes of innate immunity, have traditionally been considered a homogeneous cell population. Nevertheless, recent evidence has ...

We study low-density neutrophils within peripheral blood mononuclear cells to characterize their functions in order to define their role in different diseases. Low-density neutrophils are rare in healthy blood, but they markedly increase in diseases such as systemic lupus erythematosus and cancer. To begin, add 10 units per milliliter of heparin as an anticoagulant into a 15-milliliter conical centrifuge tube.Then add two milliliters of 6%dextran T500 in PBS. Obtain 10 milliliters of blood from a healthy adult volunteer by venipuncture. In another fresh 15-milliliter centrifuge tube, add five milliliters of density gradient medium.Carefully pipette out the plasma without touching the erythrocytes and layer it on top of the medium to form two separate phases. Centrifuge the tube at 516 g for 20 minutes at four degrees Celsius. To isolate PBMCs, aspirate and discard the plasma above the PBMC band without disturbing the cells.Collect the mononuclear cell band between the plasma and the medium, minimizing collection of the medium. Add 20 milliliters of PBS to the tube containing PBMCs, then centrifuge at 400 g for five minutes at four degrees Celsius. Carefully aspirate out the supernatant and scrape the tube to separate the pellet.Add 10 milliliters of cold PBS to resuspend the cells. To isolate neutrophils, scrape the tube to separate the cells after pipetting out the medium. Then add 10 milliliters of cold PBS.Now transfer the cells into a fresh 50-milliliter conical centrifuge tube and centrifuge. Aspirate the supernatant and scrape the tube again. Now pipette 10 milliliters of cold hypotonic solution into the tube and mix gently.Mix gently for exactly one minute. Quickly add 10 milliliters of cold hypertonic solution to make the solution isotonic. Then count the neutrophils using a Neubauer chamber and ensure the purity is greater than 95%Centrifuge the suspension to obtain a cell pellet.Then resuspend the pellet in cold PBS. Centrifuge the peripheral blood mononuclear cells at 400 g for five minutes at four degrees Celsius. After removing the supernatant, resuspend the cells in 120 microliters of cold wash buffer.Then pipette 35 microliters of CD66b magnetic microbeads to the cell suspension. Incubate the mixture in the dark at four degrees Celsius for 30 minutes. Now pipette one milliliter of cold wash buffer to the tube.Centrifuge the tube at 400 g for three minutes. Scrape the tube to separate the cell pellet after removing the supernatant. And resuspending the cells in one milliliter of wash buffer.Place a magnetic separation column onto a magnet. Add 0.5 milliliters of wash buffer to the column, allowing it to pass through completely. Transfer one milliliter of the resuspended cells onto the column and let the buffer pass through drop by drop.After washing, transfer the column into a microcentrifuge tube. Then pipette one milliliter of wash buffer into the column. Now insert the plunger on top of the column.Gently apply pressure to elute the cells. Then remove the plunger and place the column onto a new microcentrifuge tube. Add another milliliter of wash buffer to the column.Then insert the plunger again and gently apply pressure to elute the remaining cells. Centrifuge both microcentrifuge tubes at 800 g for three minutes. Resuspend the cell pellets from both tubes into one milliliter of cold PBS.Keep the cell suspension on ice. Resuspend the purified cells in labeling buffer made of 1%fetal bovine serum in PBS. Transfer 250 microliters of the suspension into a 1.5-milliliter microcentrifuge tube.Add the corresponding antibodies against neutrophil membrane molecules to the tube. Then incubate the cells for 30 minutes at four degrees Celsius, protected from light. Now pipette one milliliter of PBS to the tube.Spin the tube in a microcentrifuge at 800 g for three minutes. Aspirate out the supernatant. Tap the tube to break the pellet.And resuspend the cells in 0.5 milliliters of 1%paraformaldehyde. Then keep the cells at four degrees Celsius, protected from light until analysis by flow cytometry. Analyze the samples using flow cytometry, capturing 10, 000 events per sample.Low-density neutrophils in healthy individuals represented approximately 5%of the peripheral blood mononuclear cells, while the described magnetic isolation protocol yielded low-density neutrophils at about 98%recovery. Neutrophils express the membrane markers CD10, CD11b, CD15, CD62L, and CD66b. Magnetically purified low-density neutrophils expressed the same membrane markers as neutrophils.Purified low-density neutrophils exhibited a multilobulated nucleus and were similar in size to neutrophils. Both neutrophils and low-density neutrophils generated reactive oxygen species in response to PMA stimulation, with low-density neutrophils producing higher levels than neutrophils. Low-density neutrophils released neutrophil extracellular traps in response to PMA stimulation as evidenced by colocalization of DNA with elastase and with citrulline.Both purified neutrophils and low-density neutrophils released NETs with similar kinetics and amounts after PMA treatment. Our protocol provides a fast and reproducible way to obtain highly pure and functional low-density neutrophils. We address the lack of a standardized, time-efficient protocol for isolation of low-density neutrophils from human blood.This protocol does not require any special training for complex instruments, and it's also more economical and faster than FACS.

rapid-magnetic-microbead-method-for-efficient-purification-low

Research

JoVE Journal

Immunology and Infection

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 Isolation Protocol

The video explains a way to purify B-cells by immunomagnetic targeting and separation from peripheral blood mononuclear cells suspension. The method involve labeling unwanted cell types with specific antibodies or ligands (linked to magnetic particles) targeting specific cell surface proteins, to be depleted from the heterogenous cell suspension by magnetic separation.

Negative Immunomagnetic Selection: A Method to Purify B-cells from Peripheral Blood Mononuclear Cells

In this video, we describe the procedure for isolating low-density neutrophils, or LDNs, from the peritoneal lavage fluid of a postoperative human patient. The LDNs thus obtained can be used in further downstream experiments to understand their role in tumor growth and progression.

Peritoneal Low-density Neutrophil Isolation: A Technique to Obtain Low-density Neutrophils from Peritoneal Lavage Fluid Using Magnetic Activated Cell Sorting

We describe a protocol for isolation and purification of neutrophils from mouse bone marrow by density gradient centrifugation and for neutrophil labeling using CellTracker dyes. This represents a simple, fast, reproducible and economical method for obtaining large numbers of neutrophils for downstream functional studies or adoptive transfer and tracking experiments.

Isolation, Purification and Labeling of Mouse Bone Marrow Neutrophils for Functional Studies and Adoptive Transfer Experiments

Neutrophils play an important role not only in host defense against invading microorganisms, but are also involved in the immune surveillance of tumor cells. Here, we describe techniques related to the isolation of neutrophils with anti-tumor properties and methods for monitoring anti-tumor neutrophil function in vitro and in vivo.

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

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.

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Mutations in the leucine rich repeat kinase 2 gene (LRRK2) cause hereditary Parkinson’s disease. We have developed an easy and robust method for assessing LRRK2-controlled phosphorylation of Rab10 in human peripheral blood neutrophils. This may help identify individuals with increased LRRK2 kinase pathway activity.

Human Peripheral Blood Neutrophil Isolation for Interrogating the Parkinson's Associated LRRK2 Kinase Pathway by Assessing Rab10 Phosphorylation

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.

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

This research outlines two techniques for isolating abundant neutrophil extracellular traps (NETs) from rat bone marrow. One method combines a commercial neutrophil isolation kit with density gradient centrifugation, while the other employs only density gradient centrifugation. Both approaches yield functional NETs surpassing those from peripheral blood neutrophils.

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

This protocol details a high-throughput automation-compatible method to isolate human peripheral blood mononuclear cells for biobanking and other purposes.

Isolating Human Peripheral Blood Mononuclear Cells from Buffy Coats via High Throughput Immunomagnetic Bead Separation

Here, we provide a reliable approach for isolating low- and normal-density neutrophils from whole blood using magnetic isolation (negative selection) and discontinuous density gradient medium. It ensures untouched isolation of high-purity cells (≥93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.

The Isolation and Characterization of Low- and Normal- Density Neutrophils from Whole Blood

Learn to isolate pure low-density neutrophils using density-gradient centrifugation and magnetic microbead separation. This efficient method enables functional studies of LDN in immunology and disease research.

Rapid Magnetic-microbead Method for Efficient Purification of Low-density Neutrophils

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