Mouse neutrophil isolation kit

Manufactured by Miltenyi Biotec

Sourced in Germany, United States

The Mouse Neutrophil Isolation Kit is a laboratory product designed to isolate neutrophils from mouse blood samples. The kit utilizes a specific process to separate and purify neutrophils from the sample, allowing for further analysis and investigation of these immune cells.

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Lab products found in correlation

Ls column, by Miltenyi Biotec (4 mentions) Midimacs separator, by Miltenyi Biotec (4 mentions) Easysep direct human neutrophil isolation kit, by STEMCELL (2 mentions) Mouse plasmacytoid dendritic cell isolation kit 2, by Miltenyi Biotec (2 mentions) Cytoselect leukocyte transmigration assay, by Cell Biolabs (2 mentions) Infinite m nano plate reader, by Tecan (2 mentions) H2dcfda, by Thermo Fisher Scientific (2 mentions) Anti cd3, by Thermo Fisher Scientific (1 mentions) Anti cd28, by Thermo Fisher Scientific (1 mentions) Recombinant mouse gm csf, by Thermo Fisher Scientific (1 mentions) Anti cd80, by R&D Systems (1 mentions) Mouse pan t cell isolation kit, by Miltenyi Biotec (1 mentions) Mouse il 2, by Thermo Fisher Scientific (1 mentions) Anti pd 1 neutralizing antibody, by BioXCell (1 mentions) Nebnext ultra 2 rna library prep with sample purification beads, by New England Biolabs (1 mentions) Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions) Novaseq 6000, by Illumina (1 mentions) Rneasy plus mini kit, by Qiagen (1 mentions) Easysep magnet, by STEMCELL (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)

36 protocols using mouse neutrophil isolation kit

Neutrophil-T Cell Co-culture Protocol

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Neutrophils from female C57BL/6 mice (8 weeks old) were derived from the bone marrow using the mouse Neutrophil Isolation Kit (Miltenyi Biotec), and were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 °C with 5% CO2. Neutrophils were stimulated with mouse recombinant GM-CSF (100 ng/mL, Peprotech) for 24 h, in the presence or absence of anti-CD80 (2 µg/mL, R&D System) and anti-CD86 neutralizing antibody (2 µg/mL, R&D System).
T cells for MC38-T cell co-culture were isolated from the spleens of mice with mouse pan-T cell isolation kits (Miltenyi Biotec) according to the manufacturer’s instructions. T cells were cultured in RPMI 1640 supplemented with 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37 °C with 5% CO2. Before experiments, T cells were prestimulated with mouse IL-2 (200 U/ml, Peprotech), anti-CD3 (1 µg/mL,Peprotech) and anti-CD28 (1 µg/mL, Peprotech) in the presence of MC38 cells at a 20:1 ratio for 48 h. T cells were also treated with anti-PD-1 neutralizing antibody (1 μg/ml, BioXcell) for 24 h. For neutrophil-T cell co-culture, T cells were co-cultured with pre-stimulated neutrophils at a 2:1 ratio in RPMI-1640 medium for another 24 h^{35 (link)}.

Sui Q., Zhang X., Chen C., Tang J., Yu J., Li W., Han K., Jiang W., Liao L., Kong L., Li Y., Hou Z., Zhou C., Zhang C., Zhang L., Xiao B., Mei W., Xu Y., Qin J., Zheng J., Pan Z, & Ding P.R. (2022). Inflammation promotes resistance to immune checkpoint inhibitors in high microsatellite instability colorectal cancer. Nature Communications, 13, 7316.

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Mouse Neutrophil RNA-seq Profiling

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Mouse BM neutrophils were enriched using a mouse neutrophil isolation kit (Miltenyi Biotec). Total RNAs were isolated using the RNeasy Plus Mini Kit (Qiagen), and 300 ng of total RNA was used to generate the RNA-seq library using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (NEB) according to the manufacturer’s instructions. Briefly, mRNAs were isolated from total RNAs with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), fragmented, and primed. First-strand cDNAs were synthesized, following by second-strand synthesis. The ends of purified double-strand cDNAs were repaired, ligated with an adaptor, and amplified with barcoded primers (NEBNext Multiplex Oligos for Illumina) using PCR. The purified final PCR product was sequenced using Illumina NovaSeq 6000. The reads were aligned to the mouse transcriptome (mm10) using a default setting of STAR (v.020201). Aligned reads were counted using HTseq (v.0.6.1p1). GSEA was performed using inflammation-related gene sets (positive regulation of inflammatory response (gene ontology [GO]: 0050729) and negative regulation of inflammatory response (GO:0050728)) obtained from the GO database. The Gene Expression Omnibus accession no. is GSE172394.

Li J., Kumari T., Barazia A., Jha V., Jeong S.Y., Olson A., Kim M., Lee B.K., Manickam V., Song Z., Clemens R., Razani B., Kim J., Dinauer M.C, & Cho J. (2021). Neutrophil DREAM promotes neutrophil recruitment in vascular inflammation. The Journal of Experimental Medicine, 219(1), e20211083.

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Isolation of Human and Murine Neutrophils

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Human peripheral neutrophils (hNeu) were isolated from buffy coats (German Red Cross, Ulm, Germany, www.drk-ulm.de) using gradient centrifugation. Murine bone marrow neutrophils (mNeu) were isolated by immunomagnetic separation using the negative depletion method with mouse neutrophil isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). The details are presented in Supporting Information Experimental Procedures.

Jiang D., Muschhammer J., Qi Y., Kügler A., De Vries J.C., Saffarzadeh M., Sindrilaru A., Beken S.V., Wlaschek M., Kluth M.A., Ganss C., Frank N.Y., Frank M.H., Preissner K.T, & Scharffetter-Kochanek K. (2016). Suppression of Neutrophil-Mediated Tissue Damage—A Novel Skill of Mesenchymal Stem Cells. Stem cells (Dayton, Ohio), 34(9), 2393-2406.

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Isolation of Murine Neutrophils and Platelets

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Mouse neutrophils were isolated from the bone marrow of the tibias and femurs using a commercially available mouse Neutrophil Isolation Kit (Miltenyi Biotec, Germany) according to the manufacturer’s instructions. The purity of the neutrophil preparations (consistently > 95%) was routinely verified with Giemsa staining, and cell viability (>97%) was verified by trypan blue exclusion assay.
Platelet isolation: anti-coagulated blood treated with citric acid-citrate-dextrose (ACD; 1:9 blood vol/vol) was obtained via cardiac puncture from isoflurane-anaesthetized mice. The sampling blood volume was approximately 1.2–1.5 ml per mouse. The samples were centrifuged at room temperature at 160 g for 10 min, and the platelet-rich plasma was collected and subsequently filtered through a sepharose 2B column equilibrated with 25 mM piperazine diethanesulfonic acid buffer (PIPES). The platelets were then centrifuged at room temperature for10 min at 650 g, harvested from the precipitate and resuspended in RPMI 1640.

Liu S., Su X., Pan P., Zhang L., Hu Y., Tan H., Wu D., Liu B., Li H., Li H., Li Y., Dai M., Li Y., Hu C, & Tsung A. (2016). Neutrophil extracellular traps are indirectly triggered by lipopolysaccharide and contribute to acute lung injury. Scientific Reports, 6, 37252.

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Isolation of mouse and human neutrophils

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Mouse neutrophils were isolated by Mouse Neutrophil Isolation Kit (Miltenyi, #130-097-658). Briefly, 50 µL of the Neutrophil Biotin-Antibody Cocktail were added to 200 µL of a cell suspension (5×10⁷ total cells in Magnetic-Activated Cell Sorting (MACS) buffer) and incubated for 15 min at 4°C. After washes with MACS buffer two times, 100 µL of Anti-Biotin MicroBeads were added to 400 µL of the cell suspension. An LS column and MidiMACS separator (Miltenyi) were applied for subsequent magnetic sorting.
Human PB-derived neutrophils were isolated by EasySep Direct Human Neutrophil Isolation Kit (STEMCELL, #19666). The isolation antibody cocktail (50 µL/1 mL whole blood) and RapidSpheres beads (50 µL/1 mL whole blood) were added to whole blood and incubated for 5 min at RT successively. Then, isolation buffer was added to the cell suspension to bring the volume to 10 mL, and the tube was placed in the EasySep Magnets (STEMCELL Technologies) for 5 min. The enriched cell suspension was collected in a new tube and incubated with RapidSpheres beads (50 µL/mL) for another 5 min. A second separation with EasySep Magnets for 5 min was performed, and the enriched cell suspension was collected for subsequent in vitro induction.

Yang C., Wang Z., Li L., Zhang Z., Jin X., Wu P., Sun S., Pan J., Su K., Jia F., Zhang L., Wang H., Yu X., Shao X., Wang K., Qiu F., Yan J, & Huang J. (2021). Aged neutrophils form mitochondria-dependent vital NETs to promote breast cancer lung metastasis. Journal for Immunotherapy of Cancer, 9(10), e002875.

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Respiratory Burst Induction in Bone-Marrow Neutrophils

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Analysis of respiratory burst induction in bone-marrow derived neutrophils (BM-PMN) was carried out as described previously [73 (link),79 (link),80 (link)]. Briefly, bone-marrow cells were isolated from tibias and femurs of WT and S100A9 KO mice under sterile conditions. Neutrophils were purified using a mouse neutrophil isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). Cells (2 x 10⁵) were suspended in 50 μl RPMI/ 10% FCS and seeded into 96-well plates. Following a 30 minute resting time at 37°C/5% CO₂, 2 mM of luminol was added to each well. Cells were incubated again for 1 hour at 37°C/5% CO₂ and cells were infected with S. pneumoniae at a multiplicity of infection (MOI) of 5 to trigger respiratory burst induction followed by monitoring emitted luminescence signals expressed as relative light units (RLU) over time using a Flx800 fluorescence/luminescence reader (BioTec Instruments, Bad Friedrichshall, Germany, KC4 software).

Ostermann L., Seeliger B., David S., Flasche C., Maus R., Reinboth M.S., Christmann M., Neumann K., Brand K., Seltmann S., Bühling F., Paton J.C., Roth J., Vogl T., Viemann D., Welte T, & Maus U.A. (2023). S100A9 is indispensable for survival of pneumococcal pneumonia in mice. PLOS Pathogens, 19(7), e1011493.

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Isolation and Culture of Murine Neutrophils and Macrophages

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Neutrophils were isolated by negative selection from bone marrow (BM) cells harvested from mouse femurs and tibias using a mouse neutrophil isolation kit (Miltenyl Biotec, Bergisch Gladbach, Germany). Cells were suspended in HBSS containing CaCl₂ and MgCl₂ (Invitrogen, Carlsbad, CA) supplemented with 0.5% BSA (Invitrogen) and 2% FBS (Hyclone, Logan, UT), and counted using a hemocytometer. The purity of neutrophils, evaluated by flow cytometry using Pacific blue-labeled anti-mouse Ly6G, consistently exceeded 90%.
BM cells were cultured on petri dishes in DMEM supplemented with 10% FBS, 25% L929-conditioned medium, and 40 ng/ml of recombinant mouse M-CSF (PeproTech, Rocky Hill, NJ). The cells were refed once with fresh medium after 4 days and cultured for 3 additional days to generate BM-derived macrophages (BMDM).

Kim V.Y., Batty A., Li J., Kirk S.G., Jin Y., Tang J., Zhang J., Rogers L.K., Deng H.X., Nelin L.D, & Liu Y. (2019). Glutathione Reductase Promotes Fungal Clearance and Suppresses Inflammation during Systemic Candida albicans Infection in Mice. Journal of immunology (Baltimore, Md. : 1950), 203(8), 2239-2251.

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Tracking Neutrophil Homing and Turnover in Obesity

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Adoptive cell transfer trial design was based on previously published methods⁷⁰. Schematic representation of the trial design can be found in Fig. 7a. Briefly, bone marrow was collected from n = 10 WT or ob/ob mice, and neutrophils were enriched using a mouse Neutrophil Isolation Kit (Miltenyi Biotec). WT neutrophils were labelled with green fluorescent CellTrace dye and ob/ob neutrophils were labelled with far red fluorescent CellTrace dye (Invitrogen). WT and ob/ob neutrophils were mixed at an equal ratio (confirmed by flow cytometry), and then 3 × 10⁶ cells were injected via tail vein into WT, ob/ob or ob/ob mice + neutralizing antibody against IL5 (1 mg kg⁻¹ body weight, dosed 3 d and 1 d prior to adoptive transfer intravenously, clone TRFK5, rat IgG1 isotype control; R&D Systems)^{67 (link)}. Blood and lungs were collected for flow cytometry after 4 h and 8 h, to evaluate homing and turnover, respectively. Adoptive transfer experiments were approved by RARC and IACUC and were performed in a manner compliant with all relevant ethical regulations regarding animal research.

Quail D.F., Olson O.C., Bhardwaj P., Walsh L.A., Akkari L., Quick M.L., Chen I.C., Wendel N., Ben-Chetrit N., Walker J., Holt P.R., Dannenberg A.J, & Joyce J.A. (2017). Obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through IL5 and GM-CSF. Nature cell biology, 19(8), 974-987.

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Isolation and Purification of Murine Dendritic Cell Subsets

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Bone marrow (BM)–derived pDCs were differentiated in vitro from the BM of WT mice by using recombinant FLT-3 ligand (BMD-FLT3 pDCs); after 7 days, they were collected and purified with B220⁺microbeads, as previously described.^{11 (link)} For isolation of pDCs, B cells, and neutrophils from the spleen, a cell suspension was obtained and subjected to purification after mechanical disruption and RBC lysis. Cells were enriched from total splenic cells by using the mouse Plasmacytoid Dendritic Cell Isolation kit II, mouse neutrophil isolation kit, or biotin anti-CD19⁺ or CD43 (Ly-48) microbreads, respectively (Miltenyi Biotech, Bergisch Gladbach, Germany). For pDCs isolated from spleens, after obtaining the CD11c⁺, plasmacytoid dendritic cell antigen 1 (PDCA-1)⁺, and B220⁺ fraction, the cells were further enriched with a FACSAria III cell sorter (BD, Franklin Lakes, NJ). Purity was greater than 95%.

Cervantes-Luevano K.E., Caronni N., Castiello M.C., Fontana E., Piperno G.M., Naseem A., Uva P., Bosticardo M., Marcovecchio G.E., Notarangelo L.D., Cicalese M.P., Aiuti A., Villa A, & Benvenuti F. (2018). Neutrophils drive type I interferon production and autoantibodies in patients with Wiskott-Aldrich syndrome. The Journal of allergy and clinical immunology, 142(5), 1605-1617.e4.

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Isolation and Stimulation of pDCs and Neutrophils

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As described previously [23 (link)], for the isolation of pDCs and neutrophils from the spleen, a cell suspension was obtained and subjected to purification after mechanical disruption and RBC lysis. Cells were enriched from total splenic cells by using the mouse plasmacytoid dendritic cell isolation kit II and mouse neutrophil isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany).
Neutrophil supernatants were obtained by means of overnight culture of 1 × 10⁶ purified splenic neutrophil in 100 μl of complete medium without stimulus. Neutrophil supernatants were harvested and clarified by means of centrifugation at 4°C (10,000g for 5 min), and aliquots were stored at -80°C until further use. For pDC stimulation, 1 : 5 dilution of neutrophil supernatant was added to 5 × 10⁵ total splenic pDCs, followed by culture for 4 hours or overnight for mRNA and ELISA analysis of IFN-α production.

Chen M., Peng J., Xie Q., Xiao N., Su X., Mei H., Lu Y., Zhou J., Dai Y., Wang S., Li C., Lin G, & Cheng L. (2019). Mesenchymal Stem Cells Alleviate Moderate-to-Severe Psoriasis by Reducing the Production of Type I Interferon (IFN-I) by Plasmacytoid Dendritic Cells (pDCs). Stem Cells International, 2019, 6961052.

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