Easysep neutrophil enrichment kit

Manufactured by STEMCELL

Sourced in Canada

The EasySep neutrophil enrichment kit is a magnetic cell separation system designed to isolate neutrophils from human whole blood or bone marrow samples. The kit utilizes a proprietary antibody cocktail and magnetic particles to negatively select for neutrophils, allowing for the rapid and gentle separation of this specific cell population.

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Lab products found in correlation

Trizol, by Thermo Fisher Scientific (4 mentions) Hetasep, by STEMCELL (2 mentions) Ficoll paque, by GE Healthcare (2 mentions) High intensity ultrasonic processor, by Ningbo Scientz Biotechnology (1 mentions) Bca kit, by Beyotime (1 mentions) Urea, by Merck Group (1 mentions) Vacutainer acd solution a, by BD (1 mentions) Vacutainer venous blood collection tubes sst, by BD (1 mentions) Vacutainer tube, by BD (1 mentions) K2edta, by BD (1 mentions) Roswell park memorial institute (rpmi), by Thermo Fisher Scientific (1 mentions) Cyquant cell proliferation assay kit, by Thermo Fisher Scientific (1 mentions) Transwell chamber, by Corning (1 mentions) Tnf α, by Thermo Fisher Scientific (1 mentions) Rpmi 1640, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions) Spectramax i3x multi mode microplate reader, by Molecular Devices (1 mentions) Ethidium homodimer 1, by Thermo Fisher Scientific (1 mentions) Te2000 e microscope, by Nikon (1 mentions) Formyl methionyl leucyl phenylalanine (fmlp), by Merck Group (1 mentions)

17 protocols using easysep neutrophil enrichment kit

Neutrophil Isolation and Proteomic Analysis

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After Ficoll-Paque gradient centrifugation
of buffy coats or peripheral
blood, followed by dextran sedimentation of granulocytes and hypotonic
lysis of erythrocytes, NEUs were isolated to reach 99.7 ± 0.2%
purity by positively removing all contaminating cells using the EasySep
neutrophil enrichment kit (StemCell Technologies, Vancouver, BC, Canada)
(Nicola Tamassia). The viability of the cells was monitored by trypan
blue staining. All samples were sonicated three times on ice using
a high-intensity ultrasonic processor (Scientz) in lysis buffer (8
M urea (Sigma), 1% Protease Inhibitor Cocktail (Calbiochem), 2 mM
EDTA(Sigma)). The remaining debris was removed by centrifugation at
12 000g at 4 °C for 10 min. Finally,
the supernatant was collected, and the protein concentration was determined
with BCA kits (Beyotime Biotechnology) according to the manufacturer’s
instructions.

Li X., Gao Y., Liu J., Xujian Q., Luo Q., Huang Z, & Li J. (2022). Validation of Serotransferrin in the Serum as Candidate Biomarkers for the Diagnosis of Pulmonary Tuberculosis by Label-Free LC/MS. ACS Omega, 7(28), 24174-24183.

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BCG-Stimulated Neutrophil-Dendritic Cell Interactions

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Bone marrow derived cells (BMDCs) were extracted from the femurs and tibias of 6–8 weeks old C57BL/6 mice (IMSR Cat numberJAX:000664, RRID: IMSR_JAX:000664). Dendritic cells (DCs) were generated by growing BMDCs in RPMI complete media (with 10% FBS, 2 mmol/L L-glutamine and 0.05 mg/mL Penicillin-Streptomycin) supplemented with 1% HEPES, 1% MEM, 50 µM β-mercaptoethanol, 0.1% sodium pyruvate and 40 ng/mL murine GM-CSF for 9 days as previously described [32 (link)]. The growth media is changed every 3 days. Neutrophils were isolated from BMDCs using the EasySep™ neutrophil enrichment kit (StemCell Technologies, Vancouver, Canada). Neutrophils and DC (5 × 10⁵) were treated with BCG at a ratio (multiplicity of infection (MOI)) of 1 cell: 5 BCG for all BCG strains (Connaught, Tice and Tokyo) for 2 h. BCG containing media were removed, remaining BCG killed with media containing 200 μg/mL gentamicin for 2 h and further incubated with fresh media with 20 ng/mL GM-CSF for 16 h. For neutrophil-DC co-culture experiments, DC (2.5 × 10⁵) were added to BCG-treated neutrophils after removal of BCG that was not internalized by neutrophils. Concentration of cells were kept at 5 × 10⁵ cells/mL. The supernatants were collected and assayed for cytokines (TNFα, IL-10 and IL-2) using ELISA.

Tham S.M., Rahmat J.N., Chiong E., Wu Q., Esuvaranathan K, & Mahendran R. (2021). Intravesical High Dose BCG Tokyo and Low Dose BCG Tokyo with GMCSF+IFN α Induce Systemic Immunity in a Murine Orthotopic Bladder Cancer Model. Biomedicines, 9(12), 1766.

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Isolation of Human Neutrophils

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To isolate human neutrophils, peripheral blood was drawn into tubes containing trisodium citrate, citric acid, and dextrose (Vacutainer ACD Solution A, BD). Autologous serum, used for culture of cells was obtained by drawing blood into BD Vacutainer™ Venous Blood Collection Tubes SST, followed by centrifugation at 2500 × g for 30 min and serum collection. All blood donors provided written informed consent. For normal human blood samples, 10mls human blood was supplemented with GM-CSF (10 ng/ml) for 30 min at 37 °C followed by addition of 30 μg FITC-IgG isotype control or FcγRIIIB (3G8)-FITC-Ova conjugate for 2 h at the indicated concentrations. Blood was then incubated with Hetasep (STEMCELL Technologies) according to manufacturer protocols to deplete red blood cells and enrich leukocytes. Neutrophils were isolated from the leukocyte-rich plasma layer using a Easysep Neutrophil enrichment kit (STEMCELL Technologies). Neutrophil purity was evaluated using CD15, CD11b, CD66b, and lineage markers (CD3, CD19, and CD56) Supplementary Tables 4 and 6.

Mysore V., Cullere X., Mears J., Rosetti F., Okubo K., Liew P.X., Zhang F., Madera-Salcedo I., Rosenbauer F., Stone R.M., Aster J.C., von Andrian U.H., Lichtman A.H., Raychaudhuri S, & Mayadas T.N. (2021). FcγR engagement reprograms neutrophils into antigen cross-presenting cells that elicit acquired anti-tumor immunity. Nature Communications, 12, 4791.

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Isolation and Purification of Circulating Neutrophils

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Circulating neutrophils were isolated by density gradient centrifugation (Ficoll‐Paque; GE Healthcare Life Sciences, Marlborough, MA) of whole blood collected in BD Vacutainer tubes with K₂EDTA (BD Biosciences, Franklin Lakes, NJ) from patients with OSCC or HDs or from buffy coats of healthy donors and further purified (approximately 99.7% purity) by negative selection using the EasySep neutrophil enrichment kit (StemCell Technologies, Vancouver, Canada) as previously described.⁷¹¹ Human samples were obtained following informed, written, consent by healthy donors, in accordance with the Declaration of Helsinki. This study was carried out in accordance with the recommendations of Ethic Committee of the Azienda Ospedaliera Universitaria Integrata di Verona (Italy).

Lonardi S., Missale F., Calza S., Bugatti M., Vescovi R., Debora B., Uppaluri R., Egloff A.M., Mattavelli D., Lombardi D., Benerini Gatta L., Marini O., Tamassia N., Gardiman E., Cassatella M.A., Scapini P., Nicolai P, & Vermi W. (2021). Tumor‐associated neutrophils (TANs) in human carcinoma‐draining lymph nodes: a novel TAN compartment. Clinical & Translational Immunology, 10(2), e1252.

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Isolation and Activation of Immune Cells

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Neutrophils were freshly prepared from mouse bone marrow using EasySep Neutrophil enrichment kit (STEMCELL technology). CD4⁺ and CD8⁺ T lymphocytes from spleen and lymph node were isolated using a negative selection method and activated by culturing them on a CD3 (10 µg/ml) antibody-coated dish in the presence of CD28 (2 µg/ml) and 10 unit/ml IL-2. F4/80 positive mouse monocytes were isolated from blood using FACS sorting.

Lim K., Hyun Y.M., Lambert-Emo K., Capece T., Bae S., Miller R., Topham D.J, & Kim M. (2015). Neutrophil trails guide influenza-specific CD8+ T cells in the airways. Science (New York, N.Y.), 349(6252), aaa4352.

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Isolation of Murine Neutrophils from Bone Marrow

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As described previously [54 (link)], mouse neutrophils were isolated from the bone marrow of mice. Mice were euthanized, femur and tibia were dissected and flushed with ice-cold 1× Hanks balanced salt solution (HBSS; supplemented with 10 nM HEPES, pH7.5, and 0.5 mM EDTA; Invitrogen) through a sterile filter (70 μm). The suspension was centrifuged at 300× g for 12 min at 4 °C. EasySep™ neutrophil enrichment kit (STEMCELL Technologies Inc., Vancouver, BC, Canada) was used to isolate neutrophils according to the manufacturer’s instructions. The isolated neutrophils were then washed once and the pelleted cells were suspended in RPMI (Invitrogen) containing 1% heat-inactivated fetal bovine serum (FBS, Invitrogen) and used immediately for neutrophil migration experiments or processed for adoptive transfer. The murine neutrophil isolation protocol routinely yields cell suspensions that are >95% pure and viable, as determined by cresyl violet and trypan blue exclusion, respectively.

Porter R.F., Szczesniak A.M., Toguri J.T., Gebremeskel S., Johnston B., Lehmann C., Fingerle J., Rothenhäusler B., Perret C., Rogers-Evans M., Kimbara A., Nettekoven M., Guba W., Grether U., Ullmer C, & Kelly M.E. (2019). Selective Cannabinoid 2 Receptor Agonists as Potential Therapeutic Drugs for the Treatment of Endotoxin-Induced Uveitis. Molecules, 24(18), 3338.

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Neutrophil Isolation and Migration Assay

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Circulating neutrophils were isolated from healthy donors by density gradient centrifugation (Ficoll-Paque; GE Healthcare Life Sciences) of whole blood and further purified by negative selection using the EasySep neutrophil enrichment kit (StemCell Technologies, Vancouver, BC, Canada) as previously described [42 (link)]. The purity of isolated neutrophils was > 99.8%, as determined by flow cytometry. Neutrophil direct migration (chemotaxis) was measured in Transwell chamber (3 μm; Corning Costar), as previously described [43 (link)]. Briefly, 100 μL of neutrophil suspensions (2 × 10⁶/mL) were added to the top chambers, whereas 600 μL of control medium or tumor-conditioned supernatants from luminal-type RT4 or basal-type 5637 UBC cell lines, either unstimulated or treated with the pro-inflammatory cytokine cocktail (TNF-a, IL6 and IL1b) for 4 or 24 h (as described above) were added to the bottom wells. After 45 min, the plates were spun, the inserts were removed, and the number of migrated cells were counted with CyQuant cell proliferation assay kit (Invitrogen SRL). Parallel samples were included to determine the signal intensity from the total cell number loaded into the Transwell inserts.

Mandelli G.E., Missale F., Bresciani D., Benerini Gatta L., Scapini P., Caveggion E., Roca E., Bugatti M., Monti M., Cristinelli L., Belotti S., Simeone C., Calza S., Melocchi L, & Vermi W. (2020). Tumor Infiltrating Neutrophils Are Enriched in Basal-Type Urothelial Bladder Cancer. Cells, 9(2), 291.

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Isolation and Purification of Neutrophils

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Buffy coats from healthy volunteers (through the courtesy of the Centro Trasfusionale, Spedali Civili di Brescia, Brescia, Italy) were used to isolate and purify neutrophils. Briefly, after Ficoll-Paque gradient centrifugation, granulocytes were isolated by dextran sedimentation and hypotonic lysis of erythrocytes yielding a neutrophil population with a purity higher than 90%, as assessed by flow cytometry using CD16/CD66b staining (Miltenyi Biotec, Bergisch Gladbach, Germany). For some experiments, to obtain higher neutrophil purity, the EasySep neutrophil enrichment kit (Stem cell Technologies) was used (>99%) [19 (link)].

Schioppa T., Nguyen H.O., Salvi V., Maugeri N., Facchinetti F., Villetti G., Civelli M., Gaudenzi C., Passari M., Sozio F., Barbazza I., Tamassia N., Cassatella M.A., Del Prete A., Bosisio D, & Tiberio L. (2022). The PDE4 Inhibitor Tanimilast Restrains the Tissue-Damaging Properties of Human Neutrophils. International Journal of Molecular Sciences, 23(9), 4982.

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Neutrophil Activation and Characterization

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10mls human blood was supplemented with GM-CSF (10 ng/ml) for 30 min at 37C followed by addition of 30μg AAC or FITC-IgG isotype control for 2 hr at 37C. Blood was then incubated with Hetasep (STEMCELL Technologies) according to manufacturer protocols to deplete red blood cells and enrich leukocytes. Neutrophils were isolated from the leukocyte-rich plasma layer using a Easysep Neutrophil enrichment kit (STEMCELL Technologies) and placed in RPMI media, which was supplemented with 10% autologous serum, penicillin/streptomycin (50 U/ml penicillin and 50 mg/ml streptomycin) and 20ng/ml GM-CSF. AAC-treated samples were additionally incubated with 10 μg/mL AAC. After 48 hours, cells were harvested using Accutase and evaluated by flow cytometry for surface markers of APCs.

Mysore V., Cullere X., Settles M.L., Ji X., Kattan M.W., Desjardins M., Durbin-Johnson B., Gilboa T., Baden L.R., Walt D.R., Lichtman A.H., Jehi L, & Mayadas T.N. (2021). Protective heterologous T cell immunity in COVID-19 induced by the trivalent MMR and Tdap vaccine antigens. Med (New York, N.y.), 2(9), 1050-1071.e7.

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Neutrophil Isolation and Activation Assay

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Primary rat neutrophils from bone marrow and blood were isolated using the EasySep Neutrophil Enrichment Kit (Stemcell Technologies) according to the manufacturer’s instructions, and maintained in RPMI-1640 (Invitrogen) containing 10% FBS. After culturing for 24 h, the enriched neutrophils (1×10⁵ per well) were treated with EP (0.3 mM) for 1 h and then exposed to TNF-α (100 ng/mL, ebioscience) for 24 h. The culture medium was collected after 24-h incubation to quantify the released MMP-9 using ELISA as described above.

Shi H., Wang H., Pu H., Shi Y., Zhang J., Zhang W., Wang G., Hu X., Leak R.K., Chen J, & Gao Y. (2014). Ethyl Pyruvate Protects against Blood–Brain Barrier Damage and Improves Long‐term Neurological Outcomes in a Rat Model of Traumatic Brain Injury. CNS Neuroscience & Therapeutics, 21(4), 374-384.

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