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Netosis assay kit

Manufactured by Cayman Chemical
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The NETosis assay kit is a laboratory tool designed to detect and quantify the formation of neutrophil extracellular traps (NETs) in biological samples. NETs are web-like structures released by neutrophils, a type of white blood cell, as part of the immune response. The kit provides the necessary components and protocols to measure NET formation, which is a key process in various physiological and pathological conditions.

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Lab products found in correlation

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26 protocols using netosis assay kit

1

Neutrophil Elastase Activity Assay

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2x106 mock or X4 HIV-1 infected HLACs were co-cultured with 1x106 PMNs for 3 and 24h. Mock HLACs co-cultured with PMNs in the presence of PMA (20nM, Sigma-Aldrich) served as a positive control. NE activity was analyzed by the NETosis Assay Kit (Cayman) according to the manufacturer‘s instructions. In brief, after the indicated incubation time the supernatant was aspirated and the cells were washed twice by the addition of 200μl prewarmed NET Assay Buffer followed by the addition of 200μl S7 Nuclease for 15min at 37°C. 200μl of the supernatant was transferred to a 96-well flat bottom plate containing 4μl of EDTA Assay Reagent solution and centrifuged for 5min at 300 g. To inactivate HIV-1, 180μl of the supernatant was transferred to a new 96-well flat bottom plate containing 20μl of 5% Triton X-100 and incubated for 10min at room temperature. For the preparation of the assay, 100μl of standard and sample were added to a new 96-well flat bottom plate, 100μl of the 1:30 diluted Elastase substrate was added, the plate was covered and incubated for 1-2h at 37°C. The cover was removed and the absorbance was analyzed at 405nm using the Infinite 200 PRO plate reader (Tecan).
Reif T., Dyckhoff G., Hohenberger R., Kolbe C.C., Gruell H., Klein F., Latz E., Stolp B, & Fackler O.T. (2021). Contact-dependent inhibition of HIV-1 replication in ex vivo human tonsil cultures by polymorphonuclear neutrophils. Cell Reports Medicine, 2(6), 100317.
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2

Quantification of NETosis Induced by F. alocis

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The Cayman Chemical NETosis assay kit, which allowed for the detection of extracellular NE present on NETs and distinguish it from the NE release due to granule exocytosis, was used to quantify NETs induced by F. alocis. Neutrophils (1 × 106 cells/condition) were seeded into a 24-well plate in NETs assay media (RPMI + 0.5% BSA + 1 M CaCl2) and incubated for 30 min at 37°C with 5% CO2 to allow cells to settle. After 30 min incubation, neutrophils were left unstimulated, or challenged with non-opsonized or opsonized F. alocis for different time points. Phagocytosis was synchronized by centrifugation at 600 g at 14 C. The assay was performed according to the manufacturer’s protocol. The ability of extracellular NE, present in the supernatants, to cleave the synthetic substrate was measured at 405 nm using a SpectraMax Soft Max Pro 5.4 spectrophotometer. NE values are expressed as mU/ml.
Armstrong C.L., Klaes C.K., Vashishta A., Lamont R.J, & Uriarte S.M. (2018). Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA. Innate Immunity, 24(4), 210-220.
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3

Quantifying Neutrophil Extracellular Traps

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NET formation was quantified using NETosis Assay Kit (Cayman Chemical #601010) following manufacturer’s instructions. Briefly, neutrophils were infected with log phase cultures of P. aeruginosa at an MOI of 1:50 in a 24 well plate. The plate was incubated at 37°C for 4h, and free elastase was washed from the well. NETs were dissolved with DNase and NET-associated elastase was collected and quantified by comparing neutrophil elastase activity to a known standard. Three biological replicates were tested in duplicate.
Pestrak M.J., Chaney S.B., Eggleston H.C., Dellos-Nolan S., Dixit S., Mathew-Steiner S.S., Roy S., Parsek M.R., Sen C.K, & Wozniak D.J. (2018). Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments. PLoS Pathogens, 14(2), e1006842.
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4

Immune Response Assays for Cell Activation

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The following were utilized in this study: G6PD activity assay kit (Abcam #ab176722), PGD activity assay kit (Abcam #ab273328), NETosis assay kit (Cayman #601010), ROS detection assay kit (Cayman #601290), mouse VEGF kit (Thermo Fisher Scientific, #KHG0111 & #MMV00), mouse TNFα kit (Thermo Fisher Scientific, #BMS223-4 & #MTA00B), Phagocytosis assay kit (Cell Biolabs #CBA-222), Mouse Syndecan-1 ELISA kit (Abcam #ab273165), ICAM-1 ELISA kit (R&D Systems #MIC100), CXCL10 ELISA kit (R&D Systems #DY466-05), IL-8 ELISA kit (MyBioSource #MBS7606860), and CXCR2 ELISA kit (MyBioSource #MBS726530). All experiment assays followed the manufacturers’ instructions as done by us previously95 (link). Treated cells were put in a serum-free medium and stimulated with LPS (1 μg/ml) for 3 hrs before assay ROS, VEGF, and TNF.
Maus K.D., Stephenson D.J., Macknight H.P., Vu N.T., Hoeferlin L.A., Kim M., Diegelmann R.F., Xie X, & Chalfant C.E. (2023). Skewing cPLA2 activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis. Science signaling, 16(793), eadd6527.
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5

Neutrophil Extracellular Trap (NET) Assay

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Purified neutrophils (1 × 106) in 0.5 mL of RPMI base cell culture medium supplemented with calcium chloride (1 M) and 1% bovine serum albumin were incubated and pre-treated with 0.25 or 25 µg/mL of DBV for 60 min in a 24-well plate. Afterwards, the cells were stimulated with PMA (100 nM) for 4 h at 37 °C. After stimulation, neutrophils were washed to remove soluble neutrophil elastase that is not NET-associated. Next, 500 µL of nuclease (15 U/mL) was added to each well, incubated for 15 min at 37 °C and then inactivated by EDTA (500 mM). Supernatants were collected, centrifuged at 1200 rpm for 10 min and analyzed by NETosis Assay Kit as a release of neutrophil elastase (Cayman Chemical, Ann Arbor, MI, USA), as indicated by the manufacturer’s instructions.
Scutera S., Sparti R., Comini S., Menotti F., Musso T., Cuffini A.M., Allizond V, & Banche G. (2023). Dalbavancin Boosts the Ability of Neutrophils to Fight Methicillin-Resistant Staphylococcus aureus. International Journal of Molecular Sciences, 24(3), 2541.
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6

Microscopy and ELISA for NET Formation

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In vitro NET formation by neutrophils was assessed by microscopy and by ELISA. For microscopy analysis, neutrophils (105 cells) from healthy donors were plated on poly-L-lysine-coated coverslips. Cells were allowed to settle for 1h, then treated with 50mM ethanol or untreated, then stimulated with 0.2nM PMA (phorbol 12-myristate 13-acetate) for 4h. Cells were then fixed in 25 formol-saline for 10min at room temperature, then stained with DAPI and visualized by fluorescent microscopy for extracellular DNA. Quantitative neutrophil elastase, which forms part of NETs, was measured using the Cayman Chemical NETosis assay kit (cat #: 601010, Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer’s specifications.
Bukong T.N., Cho Y., Iracheta-Vellve A., Saha B., Lowe P., Adejumo A., Furi I., Ambade A., Gyongyosi B., Catalano D., Kodys K, & Szabo G. (2018). Abnormal neutrophil traps and impaired efferocytosis contribute to liver injury and sepsis severity after binge alcohol use. Journal of hepatology, 69(5), 1145-1154.
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7

Neutrophil Elastase Activity Assay

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The Ficoll-Paque method was used to isolate neutrophils from blood, and subsequently 1 × 105 PMNs were cultured in each well of a 96-well-plate. The cells were then treated with 100 nm PMA, different concentrations of E44, or E44 along with MEM for 4 h. After the fluorescent DNA dye Picogreen was added and incubated with the samples for 10 min, the fluorescence value of every group was estimated with fluorescence microplate reader. Neutrophil elastase activity was measured using NETosis assay kit (Cayman Chemical) according to the manufacturer's instruction.
Peng L., Li L., He X.L., Yu J.Y., Zeng Z.J., Yang W.J., Zhang B., Zhang T.S., Cao H., Huang S.H, & Liu L.Q. (2020). Memantine Displays Antimicrobial Activity by Enhancing Escherichia coli Pathogen-Induced Formation of Neutrophil Extracellular Traps. Frontiers in Cellular and Infection Microbiology, 10, 47.
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8

NETosis Assay for Neutrophil Function

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The NETosis assay kit (Cayman) was used. Bead-sorted NDNs and LDNs were resuspended at 1x106 (link) cells/ml and left untreated or stimulated with PMA 20nM and incubated at 37°C for 2 hours. Culture supernatants were removed, and wells were washed to remove soluble elastase. After treatment with S7 nuclease to induce the release of NET-associated elastase, supernatants were collected, and elastase activity was evaluated according to manufacturer’s instructions. A detailed reagent list is reported in Supplementary Table 44.
Montaldo E., Lusito E., Bianchessi V., Caronni N., Scala S., Basso-Ricci L., Cantaffa C., Masserdotti A., Barilaro M., Barresi S., Genua M., Vittoria F.M., Barbiera G., Lazarevic D., Messina C., Xue E., Marktel S., Tresoldi C., Milani R., Ronchi P., Gattillo S., Santoleri L., Di Micco R., Ditadi A., Belfiori G., Aleotti F., Naldini M.M., Gentner B., Gardiman E., Tamassia N., Cassatella M.A., Hidalgo A., Kwok I., Ng L.G., Crippa S., Falconi M., Pettinella F., Scapini P., Naldini L., Ciceri F., Aiuti A, & Ostuni R. (2022). Cellular and transcriptional dynamics of human neutrophils at steady state and upon stress. Nature immunology, 23(10), 1470-1483.
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9

Neutrophil NET Formation Imaging

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For microscopic analysis, treated bone marrow-derived neutrophils or renal tissue-derived primary neutrophils were plated on poly l-lysine-coated cover slips. Neutrophils were incubated overnight at 4°C with antibodies against Cit-H3 (1:100) followed by goat anti-rabbit IgG for 1 hr at room temperature (15–25°C). The cells were counterstained with DAPI and visualized using fluorescence microscopy. The levels of neutrophil elastase, which forms part of NETs, were measured using the Cayman Chemical NETosis assay kit according to the manufacturer’s specifications. The levels of dsDNA, which forms another part of NETs, were measured using PicoGreen according to the manufacturer’s specifications.
Du C., Xu C., Jia P., Cai N., Zhang Z., Meng W., Chen L., Zhou Z., Wang Q., Feng R., Li J., Meng X., Huang C, & Ma T. (2024). PSTPIP2 ameliorates aristolochic acid nephropathy by suppressing interleukin-19-mediated neutrophil extracellular trap formation. eLife, 13, e89740.
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10

Quantification of NETosis Induced by F. alocis

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The Cayman Chemical NETosis assay kit, which allowed for the detection of
extracellular NE present on NETs and distinguish it from the NE release due to
granule exocytosis, was used to quantify NETs induced by F.
alocis. Neutrophils (1 × 106 cells/condition) were
seeded into a 24-well plate in NETs assay media (RPMI + 0.5% BSA + 1 M
CaCl2) and incubated for 30 min at 37℃ with 5% CO2 to
allow cells to settle. After 30 min incubation, neutrophils were left
unstimulated, or challenged with non-opsonized or opsonized F.
alocis for different time points. Phagocytosis was synchronized by
centrifugation at 600 g at 14℃. The assay was performed
according to the manufacturer’s protocol. The ability of extracellular NE,
present in the supernatants, to cleave the synthetic substrate was measured at
405 nm using a SpectraMax Soft Max Pro 5.4 spectrophotometer. NE values are
expressed as mU/ml.
Armstrong C.L., Klaes C.K., Vashishta A., Lamont R.J, & Uriarte S.M. (2018). Filifactor alocis manipulates human neutrophils affecting their ability to release neutrophil extracellular traps induced by PMA. Innate Immunity, 24(4), 210-220.
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