Name: isolation of human neutrophils from whole blood and buffy coats
Uploaded: 2021-09-17T14:30:00.000+00:00
Duration: 8 min 12 s
Description: Watch this Scientific Journal Video about Isolation of Human Neutrophils from Whole Blood and Buffy Coats at JoVE.com

This Project was supported by P01HL095489. A.Y.H was supported by T32HL066987.

NOTE: Human neutrophils were isolated from venous blood from discarded white blood cell filters obtained from the Blood Bank Lab at the Boston Children&#39;s Hospital. Blood donors were unidentifiable, and there was no interaction with living individuals or knowledge of identifiable personal information. Therefore, this work is not classified as human subjects research under the HHS human subjects regulations (45 CFR Part 46). The Boston Children&#39;s Hospital Institutional Review Board (IRB) approved the protocol.
1. Layering the gradient
<ol>
	<li>After sterilizing the buffy coat or whole blood packaging and the laminar hood, divide the blood into 50 mL tubes with 10 mL of blood in each tube.</li>
	<li>Bring the volume up to 35 mL with 5% fetal bovine serum (FBS)/Hank&#39;s Balanced Salt Solution (HBSS) to dilute the blood for a cleaner gradient. 
	NOTE: When using a leukapheresis membrane, cells can be flushed out using a 60 mL syringe and 30 mL of 5% FBS/HBSS per filter. Dilution is unnecessary when working with freshly drawn whole blood.</li>
	<li>Close the 50 mL tube lid, mix it several times by inversion, and keep it upside down to have the bottom devoid of RBCs.</li>
	<li>Add 10 mL of density gradient medium (see the Table of Materials) directly beneath the blood. Ensure that the medium and the blood do not mix and that the interface is sharp (Figure 1A). 
	NOTE: This step is crucial. Ensure that the density gradient solution is at room temperature (RT) and well-mixed before each gradient. The first milliliter of the density gradient medium needs to be carefully and steadily layered as slowly as possible. Having the electronic pipette speed set to low is recommended.&#160;</li>
	<li>Gently place the tube in a centrifuge without disturbing the gradient and spin at 400 &#215; g for 30 min at RT, making sure to disable the brake. Observe how the spun gradient separates into a top serum/plasma layer, a middle white ring of peripheral blood mononuclear cells (PBMCs), a cloudy density gradient medium layer, and a bottom pellet consisting of a white, thin neutrophil band on top of the RBCs (Figure 1B). 
	NOTE: A cloudy or opaque side of the tube after centrifugation can suggest that the cells (neutrophils) are activated and may not be suitable to use.</li>
	<li>Remove the PBMCs first by emerging the suction pipette directly into the PBMC layer. Be sure to aspirate it completely while the serum/plasma layer decreases as the ring is removed. Scrape the side of the tube where cells pelleted with the suction pipette to maximize the removal of the PMBC. Carefully remove the cloudy density gradient medium layer between the PBMC ring and the neutrophil/RBC pellet. 
	&#8203;NOTE: Scraping the pelleted cells on the side of the tube increases the purity of the isolation considerably. Be careful not to aspirate the pellet, as most neutrophils are sitting directly on top of the RBCs.</li>
</ol>
2. Sedimentation of erythrocytes (RBCs)
<ol>
	<li>Using a 10 mL pipette, transfer the neutrophil/RBC pellet into a clean tube. Do not pipette up and down. Add 5% FBS/HBSS to a final volume of 25 mL. Gently mix by inversion. 
	NOTE: Performing the RBC sedimentation after the PBMC removal improves yield and decreases activation10.</li>
	<li>Directly add 25 mL of prewarmed (37 &#176;C) 3% Dextran/0.9% NaCl/H2O into the tube containing the diluted neutrophil/RBC pellet and mix gently by inversion. Place the tube on a level, non-vibrating surface for 15 min (Figure 1C). 
	NOTE: Longer sedimentation reduces RBC contamination but also decreases the yield. Furthermore, prolonged dextran exposure may lead to neutrophil activation or cell death11.</li>
	<li>Gently bring the tube back into the hood (for sterile isolation). By only slightly immersing the pipette in the liquid, collect the top layer (~30 mL) following the liquid surface downwards. 
	NOTE: If the sedimentation produces a sharp interface between the medium and the RBCs, the RBC pellet is smaller, or if a higher yield is desired, collect up to 35 mL.</li>
	<li>Spin the tube (400 &#215; g, 10 min, RT, using low brake (3)), resulting in a red pellet with no particles floating in the media.</li>
</ol>
3. Lysis of the residual RBCs
<ol>
	<li>Gently aspirate the supernatant without disrupting the pellet.</li>
	<li>Add 25 mL of sterile ultrapure water directly into the tube and gently mix by inverting for 28 s to lyse the RBCs. Do not use a pipet to resuspend the pellet. 
	NOTE: Do not exceed 30 s as prolonged hypotonic conditions can activate and lead to neutrophil death12.</li>
	<li>Immediately add 25 mL of sterile 1.8% NaCl/H2O into the tube and gently mix by inverting to bring the solution back to isotonic conditions. 
	NOTE: The solution should be red but without turbidity.</li>
	<li>Spin down at 200 &#215; g for 3-5 min with low brake (level 3) to minimize RBCs and platelet sedimenting together with the neutrophils (Figure 1D and Figure 2)13,14. 
	NOTE: The pellet should be white with a minimal RBC layer on top, which can be gently removed while the supernatant is aspirated.</li>
	<li>Resuspend neutrophils by pipetting the culture medium (10% FBS/RPMI1640) directly on the pellet, but do not pipette up and down. Rock the tube horizontally from side to side to minimize cell activation. 
	NOTE: Cells should be kept at a concentration of ~2 &#215; 106 cells/mL, as higher density will lead to increased cell activation/death15. For the same reason, the cell pellet should be resuspended as soon as possible.</li>
	<li>If cell aggregation or clumping is observed, filter the cell suspension using a 70 &#181;m mesh to discard clumped neutrophils.</li>
</ol>
4. Determining neutrophil isolation quality
<ol>
	<li>Stain the cells with markers specific for neutrophils (CD66b, CD11b), eosinophils (CD193) (Figure 3), and an activation marker such as CD62L (Figure 4). Acquire 20,000 cells by flow cytometry. Analyze cell purity and activation using the gating strategies proposed in Figure 3 and Figure 4. 
	NOTE: The quality of the neutrophil preparation can be assessed using a 3% acetic acid-methylene blue solution to visualize the unique lobed nucleus of neutrophils.</li>
	<li>Determine cell viability using Annexin V/propidium iodide (PI) (Figure 5). 
	NOTE: Trypan blue staining can be used to evaluate cell viability.</li>
</ol>

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B., Martinelli, S., Marone, C., Niggli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiated+HL-60+cells+are+a+valid+model+system+for+the+analysis+of+human+neutrophil+migration+and+chemotaxis.">Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis.</a> International Journal of Biochemistry and Cell Biology. 34 (7), 838-854 (2002).</li><li>Blanter, M., Gouwy, M., Struyf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+neutrophil+function+in+vitro:+Cell+models+and+environmental+factors.">Studying neutrophil function in vitro: Cell models and environmental factors.</a> Journal of Inflammation Research. 14, 141-162 (2021).</li><li>Kambara, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gasdermin+D+exerts+anti-inflammatory+effects+by+promoting+neutrophil+death.">Gasdermin D exerts anti-inflammatory effects by promoting neutrophil death.</a> Cell Reports. 22 (11), 2924-2936 (2018).</li><li>Loison, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteinase+3-dependent+caspase-3+cleavage+modulates+neutrophil+death+and+inflammation.">Proteinase 3-dependent caspase-3 cleavage modulates neutrophil death and inflammation.</a> Journal of Clinical Investigation. 124 (10), 4445-4458 (2014).</li><li>Siemsen, D. 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The authors declare that the research was conducted in the absence of any conflict of interest.

Due to the short life span, terminal differentiation status, and lytic content of neutrophils, studying these cells has always been a challenge. Apart from utilizing mouse models or cells from patient cohorts, cell lines are useful tools to help study neutrophil biology16. However, neutrophil-like cell lines cannot completely reiterate all aspects of neutrophil biology, adding an extra layer of difficulty in studying these cells. The most commonly used in vitro model is the HL-60 cell line, which can be differentiated into neutrophil-like cells by treatment with dimethylsulfoxide or retinoic acid17,18. Although these cells are useful in the study of migration and respiratory burst, they are not suitable for studying the microbiocidal activity of neutrophils. Other cell lines exist (PLB-98, NB4), and they are also associated with their set of limitations19. 
It is pivotal to validate with primary human neutrophils observations made with mouse disease models and cell lines. Neutrophils cannot be cryopreserved efficiently and thus are often freshly isolated from whole blood or buffy coats obtained from donors and immediately processed. Once isolated, the cells start to undergo a complex form of spontaneous death, regulated by oxidation, cytoplasmic caspases, and proteases found in neutrophil granules20,21. Improper isolation methods or techniques can lead to the activation of neutrophils, only accelerating cell demise. It is imperative to have a reliable and consistent method to obtain pure and high-quality neutrophils from donors. 
There have been many methods published on human neutrophil isolation10,22. They mainly fall into two categories, with some shared strategies. The first category is antibody-based, being either through positive or negative selection. Positive selection would label neutrophils directly, therefore providing a highly pure cell population, although it also leads to rapid cell activation, cell death, as well as unwanted tagging of neutrophils23. Negative selection, though leaving the cells unlabeled and giving a very pure population, accelerated neutrophil death, although the precise mechanism is unknown (Figure 5). Whether gene or protein expression is also altered after positive or negative selection needs to be further investigated. Moreover, due to the amount of antibody needed to deplete the other types of cells, these methods cannot output large amounts of neutrophils. However, antibody-based assays can still be used for short-term culture and experiments on a smaller scale and are methods of choice for experiments requiring very high cell purity, such as gene and protein expression studies. 
The second type of isolation method is gradient- and density-based. It usually involves Percoll, Ficoll-Paque, or other polysaccharide/polyvinylpyrrolidone components and utilizes centrifugation force to separate different types of blood cells based on cell density. These methods often are complemented with the sedimentation of red blood cells by dextran. These methods can handle larger scales of starting material and can achieve high purity as well. One caveat of density-based isolation is the inefficient separation of other far less abundant granulocytes (mostly eosinophils) from neutrophils, and it is, therefore, the major limitation of the protocol presented, as even the presence of small cell contamination can affect neutrophil response24.
Presented here is a summarized method based on gradient isolation, refining previous methods10,22. We utilize the current understating of neutrophil specificities to reliably isolate pure human neutrophils with limited residual platelet and RBCs, preventing neutrophil activation and accelerated death. The most crucial step is the layering of the gradient, which is much more effectively obtained by adding the density gradient medium underneath the blood to obtain a sharp interface. A quick examination of the PBMC ring and the gradient after centrifugation can reveal possible contamination, activation, and low yields. When working with a leukapheresis membrane or buffy coat, diluting the blood is important as excessive cell density would lead to cell aggregation, leading to impurities and cell activation. 
This protocol should be completed within 2 h to ensure cell freshness, and steps involving the density gradient medium, dextran, and lysis be done immediately, as exposure to these solutions can alter neutrophils. With this protocol, the expected yield of neutrophils is at least ~10 million/10 mL of whole blood and at least ~60 million/10 mL of buffy coat. Evaluation of isolation quality should be done as follows: activated neutrophils should be less than 10%, lymphocyte contamination lower than 5%, minimal eosinophil (same side scatter but lower forward scatter population), and cell viability should be over 90%. Lower purity can result from improper layering or storage of the density gradient medium or due to the quality and freshness of the starting blood product.

The innate immune system is comprised of many cell types that maintain immune homeostasis and pathogen clearance along with many other physiological functions. Neutrophils comprise the largest pool of white blood cells in human circulation1. Most mature neutrophils are stored in the bone marrow, which is the site of generation for new neutrophils, also called granulopoiesis. In the bone marrow, granulocyte progenitors exit the cell cycle and terminally differentiate, acquiring their characteristic segmented nuclei and granules2. Under inflammatory conditions, in response to chemokines, cytokines, and damage-associated and pathogen-associated molecular patterns, neutrophils are mobilized from the bloodstream and out of the bone marrow to perform a wide array of functions. These include cytokine secretion, direct phagocytosis of the pathogen, the release of reactive oxygen species, degranulation of antimicrobial proteins, and formation of neutrophil extracellular traps.
The molecules used by neutrophils to fight infection are toxic to the microbes and the host. Thus, the life span and the proper removal of aging/dying neutrophils are highly regulated, and they have a limited life span in circulation (&#60;48 h)3. Due to this short survival, the human body produces on average 100 billion new neutrophils&#160;every day to maintain population homeostasis4. Emergency granulopoiesis can further increase the release of neutrophils, both mature and immature, in the blood during inflammation and infection5. The importance of neutrophils in the innate immune response is highlighted by patients with acquired or congenital neutropenia, who are susceptible to bacterial and fungal infections6.
Many challenges arise when studying neutrophil biology and their roles in the immune response due to their nature, including their short survival and cytotoxic content. Neutrophil-like cell lines have been commonly differentiated from human promyelocytic leukemia HL-60 cells and PLB-985 cells7,8. Although they can display neutrophil-like morphology and perform chemotaxis, these cell lines cannot fully recapitulate the biology of neutrophils. In vitro assays using these cell lines are also unable to recapitulate in vivo experiments. Furthermore, the differentiation of these cells needs to be induced and could be adversely affected by gene manipulation before differentiation.
Recently, methods have been developed to circumvent these issues by using inducible promoters to modulate gene expression post differentiation in HL-60 cells9. Even with such tools, primary human PMNs are required to validate targets using pharmacological approaches. Thus, it is imperative to obtain pure and inactivated neutrophils isolated from blood to validate the findings of cell line and animal models. This paper presents a revised PMN isolation protocol in which the pros and cons of current methods were evaluated10. A combination was devised consisting of gradient centrifugation to separate PMNs from other immune cells, short dextran-based sedimentation to remove the bulk of RBCs, rapid residual RBC lysis via osmotic pressure, and low-speed centrifugation to remove platelet contamination.

<table><tbody><tr><td>3% Acetic Acid with Methylene Blue</td><td>Stemcell technologies</td><td>#07060</td><td></td></tr><tr><td>Attune NxT</td><td>invitrogen</td><td>A24858</td><td>FACS analyzer</td></tr><tr><td>Cd11b-PE</td><td>biolegend</td><td>301305</td><td></td></tr><tr><td>CD14-BV421</td><td>biolegend</td><td>367144</td><td></td></tr><tr><td>CD193-BV605</td><td>biolegend</td><td>310716</td><td></td></tr><tr><td>CD19-PerCP</td><td>biolegend</td><td>302228</td><td></td></tr><tr><td>CD3-PerCP</td><td>biolegend</td><td>300326</td><td></td></tr><tr><td>CD41-APC</td><td>biolegend</td><td>303710</td><td></td></tr><tr><td>Cd45-APC</td><td>biolegend</td><td>103111</td><td></td></tr><tr><td>CD62L-PE</td><td>biolegend</td><td>304802</td><td></td></tr><tr><td>CD66b-FITC</td><td>biolegend</td><td>305103</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>22625101</td><td>Centrifuge</td></tr><tr><td>Dextran</td><td>Fisher</td><td>BP1580</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>R&amp;amp;D systems</td><td>S11150H</td><td>complement inactivation of FBS is recommended</td></tr><tr><td>FITC Annexin V Apoptosis Detection Kit I</td><td>BD</td><td>556547</td><td></td></tr><tr><td>Hanks balanced salt solution (-CaCl&lt;sub&gt;2&lt;/sub&gt;), (-MgCl&lt;sub&gt;2&lt;/sub&gt;) (-MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Gibco</td><td>14175-095</td><td>HBSS without Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; is advised&amp;nbsp; as they have been shown to lead to neutrohpil activation</td></tr><tr><td>Lymphoprep</td><td>Stemcell technologies</td><td>#07801</td><td>density gradient medium</td></tr><tr><td>MACSxpress Whole Blood Neutrophil Isolation Kit, human</td><td>Miltenyi</td><td>130-104-434</td><td></td></tr><tr><td>RPMI-1640</td><td>Gibco</td><td>11875093</td><td></td></tr><tr><td>Sodium chloride</td><td>sigma</td><td>71376</td><td></td></tr><tr><td>Trypan Blue Solution, 0.4%</td><td>Thermofisher</td><td>15250061</td><td></td></tr><tr><td>ultrapure water</td><td>KD medical</td><td>RGF-3410</td><td></td></tr></tbody></table>

isolation of human neutrophils from whole blood and buffy coats

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells recruited at the site of inflammation via rapid extravasation through vessels. There, neutrophils perform an array of functions to kill invading pathogens and mediate immune signaling. Freshly purified neutrophils from human blood are the model of choice for study, as no cell line fully replicates PMN functions and biology. However, neutrophils are short-lived, terminally differentiated cells and are highly susceptible to activation in response to physical (temperature, centrifugation speed) and biological (endotoxin, chemo- and cytokines) stimuli. Therefore, it is crucial to follow a standardized, reliable, and fast method to obtain pure and non-activated cells. This protocol presents an updated protocol combining density gradient centrifugation, red blood cell (RBC) sedimentation, and RBC lysis to obtain high PMN purity and minimize cell activation. Furthermore, methods to assess neutrophil isolation quality, viability, and purity are also discussed.

When using density gradient to purify neutrophils, it is critical for the interface between the blood and the density gradient medium to be as sharp as possible, and that a distinct layer separation remains after centrifugation (step 1.4). Following RBC lysis, the buffer should be a clear red and not turbid (step 3.3). If the preparation is cloudy, a second round of lysis (step 3) may be required, although this may affect neutrophil survival (Figure 1). Following lysis, low-speed centrifugation (200 &#215; g) is recommended when purity is prioritized, as it reduces platelet contamination considerably. However, high-speed centrifugation (400 &#215; g) increases the yield at the expense of purity (step 3.4, Figure 2). After neutrophil isolation, fluorescence-activated cell sorting can be used to assess isolation purity (step 4.1) and should be chosen over microscopy. Although the FSC/SSC distribution of cells alone provides an estimate of the cell isolation quality (Figure 3A), the use of specific cell markers should be preferred. In this case, the most common contaminating cell populations are stained with specific antibodies together with CD66b, specifically expressed on granulocytes. CD45 staining is used to distinguish leukocytes (CD45+) and red blood cells and platelets (CD45-).
Other contaminants include lymphocytes (CD3+ or CD19+, Figure 3F), monocytes (CD14+, Figure 3D), and eosinophils (CD193+, Figure 3G). CD11b is an integrin expressed on the myeloid lineage; neutrophils and monocytes are CD11b+, whereas lymphocytes are CD11b- (Figure 3C). As neutrophil activation can affect downstream experiments, the expression of CD62L should be assessed; neutrophils become CD62L- once activated (step 4.1). The peptide fMLP can be used as a positive control for CD62L shedding (Figure 4). It is also important to evaluate the health of neutrophils before performing assays; neutrophils have a relatively short half-life, and activation can further shorten it (step 4.2). A standard Annexin V and PI staining can give information on the live/dead status of the neutrophil culture at designated times (Figure 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62837/62837fig01.jpg" /> 
Figure 1: Density gradient medium-based separation of granulocytes. (A) Before and (B) after centrifugation. Note the sharp interface between the blood and the density gradient medium layers. (C) Sedimentation of the pellet in B resuspended (1:1) in 5% FBS/HBSS and 3% Dextran-0.9% NaCl. (D) PMN pellet after lysis of the residual RBCs in the supernatant of (C) with H2O. Abbreviations: PBMC = peripheral blood mononuclear cell; PMN = neutrophil; RBC = red blood cell; FBS = fetal bovine serum; HBSS = Hank&#39;s Balanced Salt Solution. <a href="https://www.jove.com/files/ftp_upload/62837/62837fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62837/62837fig02.jpg" /> 
Figure 2: Spinning at lower speeds after RBC lysis decreased platelet contamination. After lysis of RBCs with H2O, cells were spun down at 200 &#215; g (A) or 400 &#215; g (B). Neutrophils were stained, and flow cytometry analysis was performed, as described in Figure 3, with the addition of anti-CD41 to label the platelets. Abbreviations: RBC = red blood cell; CD41 = cluster of differentiation 41; SSC-A = side scatter-Area. <a href="https://www.jove.com/files/ftp_upload/62837/62837fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62837/62837fig03.jpg" /> 
Figure 3: Assessment of neutrophils isolated from buffy coat. Isolated neutrophils were stained with anti-CD66b, anti-CD11b, anti-CD14, and anti-CD193 following a standard protocol. Single cells (B) were gated from total cells (A). (C) CD11b+ cells were gated from single cells. (D) Dot plot showing neutrophils (CD66b+, CD14 low/-) and a very low monocyte contamination (CD66b-, CD14+, Q1). (E) CD66b- and CD66b+ cells were gated. (F) CD66b- cells were positive for lymphocyte markers (CD3, CD19). (G) Expression of CD11b and CD193 in CD66b+ cells, showing distinct neutrophil (CD66b+, CD11b+, CD193-) and eosinophil (CD66b+, CD11b-, CD193+) populations. In the representative result shown here, neutrophil purity is ~93% with ~3.7% lymphocyte and ~3.7% eosinophil contamination. (H) Quantification of neutrophil purity after purification. Data are compiled from 5 individual trials and presented as mean &#177; SD. Abbreviations: CD = cluster of differentiation; SSC-A = SSC-A = side scatter-Area; FSC-A = forward scatter-Area. <a href="https://www.jove.com/files/ftp_upload/62837/62837fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62837/62837fig04.jpg" /> 
Figure 4: Gradient purification did not cause CD62L shedding. (A-B) Control (A) or fMLP-stimulated neutrophils (B) (1 mM fMLP for 15 min at 37 &#176;C) were stained with neutrophil markers and CD62L. (C) Fluorescent mean intensity for CD62L is decreased in fMLP-treated cells, indicating CD62L shedding and neutrophil activation. Abbreviations: CD26L = L-selectin; fMLP = N-Formylmethionyl-leucyl-phenylalanine; SSC-A = SSC-A = side scatter-Area; FSC-A = forward scatter-Area; PE = phycoerythrin. <a href="https://www.jove.com/files/ftp_upload/62837/62837fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62837/62837fig05.jpg" /> 
Figure 5: Spontaneous death of neutrophils purified by density gradient or neutrophil isolation kit. Neutrophils were purified with density gradient (A and B) or commercial microbeads (C and D) and cultured for 0 h (A and C) or 24 h (B and D) in RPMI-10% FCS. Cells were stained using Annexin V and PI at respective time points following a standard protocol. (E) Quantification of purified neutrophil spontaneous death. n=5, mean &#177; SD. Abbreviations: SSC-A = SSC-A = side scatter-Area; FSC-A = forward scatter-Area; PI = propidium iodide; FCS = fetal calf serum; FITC = fluorescein isothiocyanate; AV5 = Annexin V. <a href="https://www.jove.com/files/ftp_upload/62837/62837fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Human Neutrophils from Whole Blood and Buffy Coats at JoVE.com

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

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.

Neutrophils (PMNs) are the most abundant leukocytes in human circulation, ranging from 40 to 70% of total blood leukocytes. They are the first cells ...

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17.9K Views.  Harvard Medical School, Department of Lab Medicine, Children’s Hospital Boston, and Dana-Farber/Harvard Cancer Center. 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.

Video: Isolation of Human Neutrophils from Whole Blood and Buffy Coats

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 ...

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions.
In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves.
A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained.
We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs

Simplified Human Neutrophil Extracellular Traps NETs Isolation and Handling

In the following protocol, we describe a very simple way to isolate Neutrophil Extracellular traps (NETs) from human whole blood using readily available reagents. We then demonstrate how the isolated NETs can be used in an in vitro adhesion assay with cancer cells.

Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil&#8217;s antimicrobial armamentarium. Apart from their role ...

Immunology and Infection

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or na&#239;ve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a &#62; 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function.

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.

Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading ...

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

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

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.

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst leukocytes and in their non-activated state are constitutively committed to apoptosis. When recruited to inflammatory sites to resolve inflammation, they produce an array of cytotoxic molecules with potent antimicrobial killing. Yet, when these powerful cytotoxic molecules are released in an uncontrolled manner they can damage surrounding tissues. In recent years however, neutrophil versatility is increasingly evidenced, by demonstrating plasticity and immunoregulatory functions. We have recently identified a new neutrophil-derived subpopulation, which develops spontaneously in standard culture conditions without the addition of cytokines/growth factors such as granulocyte colony-stimulating factor (GM-CSF)/interleukin (IL)-4. Their phagocytic abilities of neutrophil remnants largely contribute to increase their size immensely; therefore they were termed giant phagocytes (G&#981;). Unlike neutrophils, G&#981; are long lived in culture. They express the cluster of differentiation (CD) neutrophil markers CD66b/CD63/CD15/CD11b/myeloperoxidase (MPO)/neutrophil elastase (NE), and are devoid of the monocytic lineage markers CD14/CD16/CD163 and the dendritic CD1c/CD141 markers. They also take-up latex and zymosan, and respond by oxidative burst to stimulation with opsonized-zymosan and PMA. G&#981; also express the scavenger receptors CD68/CD36, and unlike neutrophils, internalize oxidized-low density lipoprotein (oxLDL). Moreover, unlike fresh neutrophils, or cultured monocytes, they respond to oxLDL uptake by increased reactive oxygen species (ROS) production. Additionally, these phagocytes contain microtubule-associated protein-1 light chain 3B (LC3B) coated vacuoles, indicating the activation of autophagy. Using specific inhibitors it is evident that both phagocytosis and autophagy are prerequisites for their development and likely NADPH oxidase dependent ROS. We describe here a method for the preparation of this new subpopulation of long-lived, neutrophil-derived phagocytic cells in culture, their identification and their currently known characteristics. This protocol is essential for obtaining and characterizing G&#981; in order to further investigate their significance and functions.

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

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.

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

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

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single population, as has been done historically. In particular, low-density and normal-density neutrophils (LDNs, NDNs) have been shown to have functionally and metabolically distinct profiles, a factor that must be considered when publishing neutrophil research. Here, we present a modified method for the untouched isolation and separation of LDNs and NDNs from whole blood.
The density gradient medium (1.135 g/mL) is combined at 9:10 with 10x PBS. Specific density gradients of 55%, 70%, and 81% are subsequently made by combining the 100% density gradient medium with 1x phosphate-buffered saline (PBS). Neutrophils isolated from 12 mL of peripheral whole blood obtained from consented donors using a negative selection-based magnetic isolation kit are resuspended in the 55% fraction. A volume of 3 mL of the 81% and 70% fractions is layered into a 15 mL tube, followed by the 55% fraction containing total neutrophils. The density gradients are then centrifuged at 720 x&#160;g for 30 min. Two distinct bands are obtained at the 55%/70% interface (LDNs) and 70%/81% interface (NDNs). The cells are carefully pipetted into separate tubes and washed using PBS. The purity of the isolated fractions is determined using flow cytometry. Both LDNs and NDNs were defined as CD14lo CD15+ SSChi by flow cytometry. Isolation purity was calculated at &#8805;93% of viable cells for both types.
This method provides a reliable and efficient approach for separating LDN and NDNs from peripheral blood, ensuring high purity and viability of the isolated cells. Enhancing the precision of neutrophil isolation facilitates more accurate downstream analyses of these distinct neutrophil subpopulations. These are critical for advancing our understanding of neutrophil heterogeneity and its implications in various physiological and pathological contexts.

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 (&#8805;93%), facilitating accurate downstream analysis of neutrophil subpopulations, crucial for understanding their roles in health and disease.

Emerging research shows that the circulating neutrophil population in humans consists of diverse subtypes and should not be studied as a single ...

Efficient Rat Bone Marrow Neutrophil Extracellular Trap Isolation

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone marrow. This effort arose due to limitations associated with the traditional method of extracting NETs from peripheral blood, mainly due to the scarcity of available neutrophils for isolation. The study revealed two distinct methodologies for obtaining rat neutrophils from bone marrow: a streamlined one-step procedure that yielded satisfactory purification levels, and a more time-intensive two-step process that exhibited enhanced purification efficiency. Importantly, both techniques yielded a substantial quantity of viable neutrophils, ranging between 50 to 100 million per rat. This efficiency mirrored the results obtained from isolating neutrophils from both human and murine sources. Significantly, neutrophils derived from rat bone marrow exhibited comparable abilities to secrete NETs when compared with neutrophils obtained from peripheral blood. However, the bone marrow-based method consistently produced notably larger quantities of both neutrophils and NETs. This approach demonstrated the potential to obtain significantly greater amounts of these cellular components for further downstream applications. Notably, these isolated NETs and neutrophils hold promise for a range of applications, spanning the realms of inflammation, infection, and autoimmune diseases.

Author Spotlight: Optimized Method for Isolating Rat Neutrophils and NETs from Bone Marrow

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.

The primary aim of this research was to develop a reliable and efficient approach for isolating neutrophil extracellular traps (NETs) from rat bone ...

High-Throughput PBMC Isolation Using Immunomagnetic Beads

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

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in long-term storage, making them an ideal cell type for many downstream research purposes, including flow cytometry, immunoassays, and genome sequencing. Typically, PBMCs are isolated via&#160;density gradient centrifugation, however, it is a low-throughput workflow that is difficult and costly to scale. This article presents a high-throughput workflow using a magnetic bead-based PBMC isolation method that is quick to implement. Total cell concentration, viability, and population distribution with PBMCs obtained using density gradient isolation were compared, and cell viability and proportion of cell types were comparable for both techniques. Isolated PBMCs demonstrated over 70% viability up to 9 days after blood collection, although yield decreased by half after 5 days compared to PBMCs processed within 24 h of collection. In summary, this article describes a PBMC protocol that utilizes a bead-based approach to adapt to a high throughput workflow and demonstrates that both manual and automated bead-based methods can increase processing capacity and provide flexibility for various budgets.

Author Spotlight: Advancements in PBMC Isolation &#8211; Enhancing Throughput and Consistency in Research

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

Peripheral blood mononuclear cells (PBMCs) are a heterogeneous population of monocytes and lymphocytes. Cryopreserved PBMCs have stable viability in ...

Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Source: Sionov, R. V., et al., <a href="https://app.jove.com/v/52933">Isolation and Characterization of Neutrophils with Anti-Tumor Properties.</a> J. Vis. Exp. (2015)
In this video, we demonstrate the isolation of cytotoxic high-density neutrophils and low-density neutrophils from the blood of a tumor-bearing mouse by discontinuous sucrose gradient centrifugation.

Source: Sionov, R. V., et al., Isolation and Characterization of Neutrophils with Anti-Tumor Properties. J. Vis. Exp. (2015)
In this video, we demonstrate ...

JoVE Encyclopedia of Experiments

Immunology

Immune Systems and Components

Cell Isolation and Culture

Isolation of Lipids from Human Blood-Derived Neutrophils by Biphasic Separation

To isolate the lipids from the human peripheral blood-derived neutrophils, take the prepared neutrophils and place them on ice. Pipette the neutrophils to a 15-milliliter screw cap glass tube with a PTFE seal, and homogenize them by shaking for one minute. Then, add 2 milliliters of methanol, followed one minute later by 1 milliliter of chloroform. After shaking for another one minute, rotate the glass tubes at room temperature and 50 RPM for 30 minutes.
Next, pellet the protein fraction by centrifuging the solution at 7 degrees Celsius and 1952 times g for 10 minutes. Carefully decant the supernatant into a new 15-milliliter glass screw cap tube leaving the protein-containing pellet behind. Store the pellet at minus 20 degrees Celsius for future quantification.
Add 1 milliliter of chloroform and wait one minute. Then, add 1 milliliter of double distilled water and invert the glass screw cap tube with the sample for 30 seconds. After centrifuging the sample as before, remove and discard the upper phase down to but not including the cloudy layer. Dry the samples in a vacuum concentrator at 60 degrees Celsius and store them at minus 20 degrees Celsius until required.

Isolation of Human Neutrophils from Whole Blood and Buffy Coats

Summary

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Chapters in this video

Neutrophil Isolation Protocol

Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling

Isolation and Characterization of Neutrophils with Anti-Tumor Properties

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

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

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

Unique Approach for Isolating Rat Bone Marrow Neutrophils with Comparable Capacity

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

Isolation of Cytotoxic Neutrophils from Tumor-Bearing Mouse Blood

Isolation of Lipids from Human Blood-Derived Neutrophils by Biphasic Separation