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Easysep human neutrophil enrichment kit

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The EasySep Human Neutrophil Enrichment Kit is a laboratory product designed to isolate and enrich human neutrophils from whole blood or bone marrow samples. The kit utilizes a magnetic separation process to selectively remove unwanted cells, leaving the desired neutrophils for further analysis or experimentation.

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Lab products found in correlation

Vacutainer, by BD (8 mentions) Histopaque 1077, by Merck Group (3 mentions) Hoechst 33342 trihydrochloride dye, by Thermo Fisher Scientific (2 mentions) Pkh26pcl, by Merck Group (2 mentions) Anti human cd49d apc, by BioLegend (2 mentions) Anti human cd66a c e alexafluor 488, by BioLegend (2 mentions) Rosettesep human cd4 t cell enrichment cocktail, by STEMCELL (2 mentions) Dynabeads flowcomp human cd3 isolation kit, by Thermo Fisher Scientific (2 mentions) Ficoll, by GE Healthcare (2 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Pkh67 green fluorescent cell linker, by Merck Group (1 mentions) T25 cell culture flasks, by BD (1 mentions) Prigrow 3 medium, by Applied Biological Materials (1 mentions) Pen strep, by Thermo Fisher Scientific (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Hoechst fluorescent dye, by Merck Group (1 mentions) Kx 21n hematology analyzer, by Sysmex (1 mentions) Iscove s modified dulbecco s medium, by Thermo Fisher Scientific (1 mentions) Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)

34 protocols using easysep human neutrophil enrichment kit

1

Neutrophil Isolation and Characterization

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Fresh human blood samples from healthy volunteers, aged 18 years and older, were purchased from Research Blood Components (Allston, MA, USA). All blood specimens from patients were obtained with informed consent according to an institutional review board (IRB) approved protocol at the Massachusetts General Hospital. Peripheral blood was drawn into heparinized-tubes (Vacutainer; Becton Dickinson, Woburn, MA, USA) and human neutrophils were isolated within 2 hours after blood collection using EasySep Human Neutrophil Enrichment Kits by following manufacturer instructions (STEMCELL Technologies, Vancouver, Canada). After isolation, neutrophils were washed using cell culture medium, and the nuclei were stained with Hoechst 33342 trihydrochloride dye (Life Technologies, Woburn, MA, USA). Stained neutrophils were then resuspended in medium at a density of 7.5 × 105 cells per mL. Two hundred microliters of cell suspension were then pipetted into each well containing the array of zymosan particle clusters and sealed with a 12 mm diameter coverslip. The purity of the neutrophils was estimated by flow cytometry (using anti-CD66b fluorescent antibodies and Hoechst nuclear stain). Less than 0.1% of the isolated neutrophils had platelets attached to their surface (quantified using anti-CD61 fluorescent antibodies - Supplementary Fig. 10.).
Reátegui E., Jalali F., Khankhel A.H., Wong E., Cho H., Lee J., Serhan C.N., Dalli J., Elliott H, & Irimia D. (2017). Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming. Nature biomedical engineering, 1, 0094.
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2

Neutrophil Chemotaxis Assay

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De-identified, fresh human blood samples from healthy volunteers, aged 18 years and older, were purchased from Research Blood Components (Alston, MA). Peripheral blood was drawn in heparinized-tubes (Vacutainer; Becton Dickinson) and human neutrophils were isolated within 2 hours after blood collection using HetaSep followed by the EasySep Human Neutrophil Enrichment Kits (STEMCELL Technologies, Vancouver, Canada). After isolation, cells were washed using cell culture medium without serum. The cell membrane was stained with red fluorescent dye (PKH26PCL, Sigma-Aldrich). The stained neutrophils were re-suspended in medium at a density of 20 × 106 cells mL–1. Ten μL of cell suspension were injected into the cell compartment and incubated at 37 °C supplied with 5% CO2 for 30 minutes. Afterwards, 10 μL of each chemokine solution was injected into its designated chemokine compartment.
Cho H., Hamza B., Wong E.A, & Irimia D. (2014). On-demand, competing gradient arrays for neutrophil chemotaxis. Lab on a chip, 14(5), 972-978.
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3

Neutrophil Isolation and Characterization

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Fresh human blood samples from healthy volunteers, aged 18 years and older, were purchased from Research Blood Components (Allston, MA, USA). All blood specimens from patients were obtained with informed consent according to an institutional review board (IRB) approved protocol at the Massachusetts General Hospital. Peripheral blood was drawn into heparinized-tubes (Vacutainer; Becton Dickinson, Woburn, MA, USA) and human neutrophils were isolated within 2 hours after blood collection using EasySep Human Neutrophil Enrichment Kits by following manufacturer instructions (STEMCELL Technologies, Vancouver, Canada). After isolation, neutrophils were washed using cell culture medium, and the nuclei were stained with Hoechst 33342 trihydrochloride dye (Life Technologies, Woburn, MA, USA). Stained neutrophils were then resuspended in medium at a density of 7.5 × 105 cells per mL. Two hundred microliters of cell suspension were then pipetted into each well containing the array of zymosan particle clusters and sealed with a 12 mm diameter coverslip. The purity of the neutrophils was estimated by flow cytometry (using anti-CD66b fluorescent antibodies and Hoechst nuclear stain). Less than 0.1% of the isolated neutrophils had platelets attached to their surface (quantified using anti-CD61 fluorescent antibodies - Supplementary Fig. 10.).
Reátegui E., Jalali F., Khankhel A.H., Wong E., Cho H., Lee J., Serhan C.N., Dalli J., Elliott H, & Irimia D. (2017). Microscale arrays for the profiling of start and stop signals coordinating human-neutrophil swarming. Nature biomedical engineering, 1, 0094.
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4

Neutrophil Isolation and Fluorescent Labeling

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Human neutrophils were isolated from fresh human blood samples within 2 h of blood collection by using EasySepHuman Neutrophil Enrichment Kits (STEMCELL Technologies) and following the manufacturer's instructions. After isolation, the neutrophils were washed using 1x PBS, and immediately mixed with 4 μL of dye solution (PKH67 Green Fluorescent Cell Linker, Sigma-Aldrich). The cell/dye mixture was incubated at room temperature for 4 min and periodically mixed by pipetting to achieve a bright, uniform, and reproducible labeling. After the incubation, the staining was stopped by adding an equal volume (1 mL) of 1% BSA in PBS and incubating for 1 min to remove excess dyes. Unbound dyes were washed by centrifugation and suspending cells in culture medium (106 cells/mL). Ten μL of the cell suspension was injected into each annular compartment. The loaded microfluidic-device were then incubated at 37°C supplied with 5% CO2 in 10 min for the cell attachment and used later in microscope.
Park J., Baik S.H., Mook-Jung I., Irimia D, & Cho H. (2019). Mimicry of Central-Peripheral Immunity in Alzheimer's Disease and Discovery of Neurodegenerative Roles in Neutrophil. Frontiers in Immunology, 10, 2231.
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5

Isolation and Culture of Microglia and Neutrophils

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Immortalized human adult microglia cells (SV40-microglia) were created by a company (ABM Inc., Montreal, Canada). The cells were plated onto T25 cell culture flasks (BD Biosciences, San Jose, CA, USA) and maintained in Prigrow III Medium (ABM Inc) supplemented with 10 % FBS (Life Technologies) and 1 % Pen/Strep (Invitrogen) in a CO2 cell culture incubator. The cell culture medium was changed every 3 days until cells were confluent.
For neutrophil isolation, human peripheral blood samples from healthy volunteers, aged 18 years and older, were collected by a company (Zen-Bio Inc., Research Triangle Park, NC, USA). Peripheral blood was drawn in a 10-mL tube containing a final concentration of 5 mM EDTA (Vacutainer; Becton Dickinson). Nucleated cells were isolated using a HetaSep gradient, followed by the EasySep Human Neutrophil Enrichment Kit (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer's protocol.
Park J., Baik S.H., Mook-Jung I., Irimia D, & Cho H. (2019). Mimicry of Central-Peripheral Immunity in Alzheimer's Disease and Discovery of Neurodegenerative Roles in Neutrophil. Frontiers in Immunology, 10, 2231.
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6

Neutrophil Isolation and Staining

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Human neutrophils were was prepared as described (Cho et al., 2013 ). Briefly, neutrophils were isolated from whole blood with HetaSep and an EasySepHuman Neutrophil Enrichment Kit (STEMCELL Technologies, Vancouver, Canada). The cell membrane was stained with red fluorescent dye (PKH26PCL, Sigma-Aldrich). The stained neutrophils were re-suspended in a culturing medium at a concentration of 20 × 106 cells·mL−1. Ten µL of cell suspension were injected into the cell compartment and incubated at 37 °C supplied with 5% CO2 for 30 minutes to recover their activities.
Baik S.H., Cha M.Y., Hyun Y.M., Cho H., Hamza B., Kim D.K., Han S.H., Choi H., Kim K.H., Moon M., Lee J., Kim M., Irimia D, & Mook-Jung I. (2014). Migration of neutrophils targeting amyloid plaques in Alzheimer’s disease mouse model. Neurobiology of aging, 35(6), 1286-1292.
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7

Neutrophil Isolation and Analysis

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Neutrophils were isolated from anticoagulated blood using a negative magnetic separation kit. (EasySep Human Neutrophil Enrichment Kit, STEMCELL Technologies Inc., Canada). Isolated cells were suspended in Hanks’ balanced salts solution (HBSS) with calcium and magnesium-containing 2% fetal bovine serum (FBS, Sigma-Aldrich Chemical Co., USA) for the spontaneous DNA release assay or suspended in lysis buffer for targeted phosphoproteomics measurement.
Surmiak M., Wawrzycka-Adamczyk K., Kosałka-Węgiel J., Włudarczyk A., Sanak M, & Musiał J. (2023). Activity of granulomatosis with polyangiitis and its correlation with mTOR phosphoproteomics in neutrophils. Frontiers in Immunology, 14, 1227369.
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8

Neutrophil Isolation and Characterization

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Neutrophils were isolated from peripheral venous blood using a Percol density gradient as described previously25 (link). A further purification step was performed using the EasySep™ Human Neutrophil Enrichment Kit (STEMCELL Technologies) as per the manufacturer’s instructions26 (link). Neutrophils were resuspended in Hanks balanced salt solution (HBSS−) without calcium and magnesium (Life Technologies) and FACS analysis was performed using Anti-Human CD49d-APC (BioLegend, 304307) and Anti-Human CD66a/c/e AlexaFluor-488 (BioLegend, 342306) antibodies to confirm purity. Prior to migration, neutrophils were stained with a CellTrace Calcein Red-Orange cell stain (Thermo Fisher) as per the manufacturer’s instructions. Stained neutrophils were washed twice in HBSS− then resuspended in HBSS plus calcium and magnesium (HBSS+). Unstained neutrophils were used if downstream analysis was to be performed (FACS/ microscopy), these were resuspended in HBSS+ prior to migration.
Deng Y., Herbert J.A., Smith C.M, & Smyth R.L. (2018). An in vitro transepithelial migration assay to evaluate the role of neutrophils in Respiratory Syncytial Virus (RSV) induced epithelial damage. Scientific Reports, 8, 6777.
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9

Neutrophil Isolation and Chemotaxis Assay

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Peripheral blood samples were collected in 3 mL tubes containing a final concentration of 5 mM ethylenediaminetetraacetic acid (EDTA, Vacutainer; Becton Dickinson) and processed within 2 h of collection.
Using standard sterile techniques, we isolated neutrophils from whole blood by use of HetaSep followed by the EasySep human neutrophil enrichment kit (Stemcell Technologies) in accordance with the manufacturer's protocol. The purity of neutrophils was assessed to be >98%, using the Sysmex KX-21N Hematology Analyzer (Sysmex America). White blood cells (WBCs) were isolated using Hetasep, followed by a 5-min spin-down cycle and washing with 1 × PBS. WBCs were stained with Hoechst fluorescent dye (32.4 μM; Sigma-Aldrich). The final aliquots of WBCs were re-suspended in Roswell Park Memorial Institute (RPMI) medium plus 10% fetal bovine serum (FBS; stock 50 mL of FBS/450 mL of RPMI; Sigma-Aldrich) at a concentration of 4,000 cells/2 μL and kept at 37°C. Cells were then immediately introduced into the microfluidic device for the chemotaxis and A. fumigatus assay. All experiments were repeated at least 3 times with neutrophils or WBCs from 3 different healthy donors.
Jones C.N., Ellett F., Robertson A.L., Forrest K.M., Judice K., Balkovec J.M., Springer M., Markmann J.F., Vyas J.M., Warren H.S, & Irimia D. (2019). Bifunctional Small Molecules Enhance Neutrophil Activities Against Aspergillus fumigatus in vivo and in vitro. Frontiers in Immunology, 10, 644.
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10

Blood Fractionation and Neutrophil Isolation

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Whole blood was collected from healthy volunteers into citrated or ethylenediaminetetraacetic acid tubes. Citrated blood was centrifuged for 10 minutes at 150g to obtain platelet-rich plasma (PRP), or 10 minutes at 2× 2000g to obtain platelet-poor plasma (PPP). The average concentration of PRP ranged from ~300 to 500 × 109 cells/L. Neutrophils were purified from the ethylenediaminetetraacetic acid tubes using the EasySep Human Neutrophil Enrichment Kit (Stemcell Technologies, USA).
Liuwantara D., Chew Y.V., Favaloro E.J., Hawkes J.M., Burns H.L., O'Connell P.J, & Hawthorne W.J. (2016). Characterizing the Mechanistic Pathways of the Instant Blood-Mediated Inflammatory Reaction in Xenogeneic Neonatal Islet Cell Transplantation. Transplantation Direct, 2(6), e77.
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