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Easysep neutrophil isolation kit

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The EasySep Neutrophil Isolation Kit is a product designed for the rapid and efficient isolation of neutrophils from whole blood or bone marrow samples. The kit utilizes a negative selection approach to isolate the target cells without the need for density gradient centrifugation.

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Lab products found in correlation

Sytox green, by Thermo Fisher Scientific (3 mentions) Female c57bl 6 mice, by Jackson ImmunoResearch (2 mentions) Reparixin, by Selleck Chemicals (1 mentions) Af647, by Thermo Fisher Scientific (1 mentions) Easy 50 magnet, by STEMCELL (1 mentions) Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Avantor (1 mentions) K2 edta tube, by BD (1 mentions)

6 protocols using easysep neutrophil isolation kit

1

Isolation of Bone Marrow-Derived Macrophages and Neutrophils

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For bone marrow derived macrophages (BMDMs), femur/tibia were harvested, flushed with PBS to collect bone marrow cells. After RBC lysis, cells were centrifuged, washed, and set up in T75 flask with complete DMEM (supplemented with 10% FBS and Penicillin/Streptomycin (100U/ml)). Murine-CSF (final concentration of 50 ng/ml) was added at days 0, 2, 4, 6 for a week until they differentiate to macrophages. For bone marrow derived neutrophils (BMDNs), RBC-lysed bone marrow cells were passed through enriched neutrophil isolation cocktail EasySep neutrophil isolation kit (STEMCELL Technologies) using a magnetic negative selection.
Cai S., Paudel S., Jin L., Ghimire L., Taylor C.M., Wakamatsu N., Bhattarai D, & Jeyaseelan S. (2020). NLRP6 Modulates Neutrophil Homeostasis in Bacterial Pneumonia-Derived Sepsis. Mucosal immunology, 14(3), 574-584.
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2

Murine Peritoneal Neutrophil Isolation

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This study was approved by the Boston Children’s Hospital Institutional Animal Care and Use Committee (protocol no. 00001290). Eight-week-old C57/BL6 female mice (Jackson Laboratories) were given 0.5 mg of synthesized MSU crystal57 in 200 µl of PBS intraperitoneally and analysed after 24 h. Neutrophils from peritoneal lavage and bone marrow were isolated by negative selection using the EasySep Neutrophil Isolation Kit (Stem Cell Technologies), according to the manufacturer’s instructions. Isolated neutrophils (2 × 106 per sample) were quenched with ice-cold 80% methanol in preparation for metabolomic studies.
Britt E.C., Lika J., Giese M.A., Schoen T.J., Seim G.L., Huang Z., Lee P.Y., Huttenlocher A, & Fan J. (2022). Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils. Nature Metabolism, 4(3), 389-403.
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3

Neutrophil Activation by COVID-19 Plasma

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Neutrophils were isolated using the EasySep Neutrophil Isolation Kit (Stemcell Technologies), counted, and resuspended in RPMI containing L-glutamine and 5% FCS. Neutrophils were exposed to plasma (10% final concentration) derived from healthy donors (n = 4) or patients with COVID-19 (n = 7) and subsequently stained for indicated activation markers. For selected subgroups, human IL-8 (MilliporeSigma, I1654), anti-human IL-8 antibody (MilliporeSigma, I2519), or reparixin (20 μM, SelleckChem, S8640) were added to either plasma or neutrophils 20 minutes before neutrophil exposure to plasma. Cells were incubated at 37°C and 5% CO2 for 1 hour, fixated using 1% PFA. Mean fluorescent intensities (MFIs) of neutrophils (singlets>size>CD15++CD16+) were assessed.
In a separate series of experiments, isolated healthy neutrophils were added to poly-L lysine coated Ibidi μ-slides and treated with plasma (20% final concentration) and/or inhibitors as described above. Neutrophil granules, released vesicles and NETS were stained using antibodies against myeloperoxidase (MPO, R&D Systems, AF3667) and neutrophil alkaline phosphatase (ALPL, MilliporeSigma, HPA008765) and secondary antibodies (anti-goat AF594, anti-rabbit AF647, 1:200, Invitrogen) along with 4′,6-diamidin-2-phenylindole (1:1000) and SytoxGreen (Thermo Fisher Scientific, S7020, 500 nM final concentration, see Supplemental Figure 4C).
Kaiser R., Leunig A., Pekayvaz K., Popp O., Joppich M., Polewka V., Escaig R., Anjum A., Hoffknecht M.L., Gold C., Brambs S., Engel A., Stockhausen S., Knottenberg V., Titova A., Haji M., Scherer C., Muenchhoff M., Hellmuth J.C., Saar K., Schubert B., Hilgendorff A., Schulz C., Kääb S., Zimmer R., Hübner N., Massberg S., Mertins P., Nicolai L, & Stark K. (2021). Self-sustaining IL-8 loops drive a prothrombotic neutrophil phenotype in severe COVID-19. JCI Insight, 6(18), e150862.
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4

Isolation of Bone Marrow-Derived Macrophages and Neutrophils

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For bone marrow derived macrophages (BMDMs), femur/tibia were harvested, flushed with PBS to collect bone marrow cells. After RBC lysis, cells were centrifuged, washed, and set up in T75 flask with complete DMEM (supplemented with 10% FBS and Penicillin/Streptomycin (100U/ml)). Murine-CSF (final concentration of 50 ng/ml) was added at days 0, 2, 4, 6 for a week until they differentiate to macrophages. For bone marrow derived neutrophils (BMDNs), RBC-lysed bone marrow cells were passed through enriched neutrophil isolation cocktail EasySep neutrophil isolation kit (STEMCELL Technologies) using a magnetic negative selection.
Cai S., Paudel S., Jin L., Ghimire L., Taylor C.M., Wakamatsu N., Bhattarai D, & Jeyaseelan S. (2020). NLRP6 Modulates Neutrophil Homeostasis in Bacterial Pneumonia-Derived Sepsis. Mucosal immunology, 14(3), 574-584.
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5

Isolation of Neutrophils from Healthy Individuals

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Blood from healthy, non-hypertensive controls without cardiovascular disease was collected into K2EDTA tubes (BD) at room temperature. Less than 1 hour after blood was collected, immunomagnetic negative bead selection was performed using EasySep Neutrophil Isolation kit (StemCell Technologies, Cambridge, Massachusetts, USA) and Easy 50 magnet (StemCell Technologies) according to manufacturer’s protocol. The typical neutrophil content (CD66b+CD16+) of the final isolated fraction is 94.0%±3.7% according to manufacturer, and our lab confirmed purity by Wright-Giemsa stain. Phosphate-buffered saline (PBS) without Ca2+/Mg2+ (Thermo Fisher Scientific, Waltham, Massachusetts, USA) supplemented with 1 mM EDTA (VWR Life Science, Radnor, Pennsylvania, USA) was added to the RapidSpheres. After isolation of neutrophils, viability >90% was confirmed via Trypan Blue Solution and cells were immediately used for chemotaxis and adhesion assays. Assays were completed within 3 hours of isolation to ensure continued viability of neutrophils.
Russell D.A., Markiewicz M, & Oates J.C. (2020). Lupus serum induces inflammatory interaction with neutrophils in human glomerular endothelial cells. Lupus Science & Medicine, 7(1), e000418.
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6

Murine Peritoneal Neutrophil Isolation

Check if the same lab product or an alternative is used in the 5 most similar protocols
This study was approved by the Boston Children's Hospital Institutional Animal Care and Use Committee (Protocol 00001290). Eight-week-old C57/BL6 female mice (Jackson Laboratories) were given 0.5 mg of synthesized MSU crystal58 in 200 μL PBS i.p. and analyzed after 24 hours. Neutrophils from peritoneal lavage and bone marrow were isolated by negative selection using the EasySep Neutrophil Isolation Kit (Stem Cell Technologies) according to manufacturer’s instructions. Isolated neutrophils (2x106 per sample) were quenched with ice-cold 80% methanol in preparation for metabolomic studies.
Britt E.C., Lika J., Giese M.A., Schoen T.J., Seim G.L., Huang Z., Lee P.Y., Huttenlocher A, & Fan J. (2022). Switching to the cyclic pentose phosphate pathway powers the oxidative burst in activated neutrophils. Nature Metabolism, 4(3), 389-403.
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