Neutrophil elastase activity assay kit

Manufactured by Abcam

The Neutrophil Elastase Activity Assay Kit is a laboratory tool used to measure the activity of the enzyme neutrophil elastase. This enzyme plays a role in the immune response and is involved in various biological processes. The kit provides the necessary reagents and protocols to quantify the enzymatic activity of neutrophil elastase in a sample.

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Neutrophil elastase (21 citations) Elastase (4 citations) Activity assay kits (2 citations)

5 protocols using neutrophil elastase activity assay kit

Quantifying Neutrophil Elastase Activity in BAL

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Neutrophil elastase (NE) activity was assessed in the BAL samples using the Neutrophil Elastase Activity Assay Kit (Abcam). BAL samples were centrifuged at 500g for 5 mins and the supernatant was collected for this assay. NE activity was assessed based on the ability of NE in BAL samples to cleave a synthetic substrate that is added to the samples, thus releasing an AFC fluorophore which was quantified using a microplate reader at Ex/Em = 380/500 nm. Measurements were performed at 10 and 20 mins to assess the change in NE activity according to the following formula: ΔRFU = (RFU₂₀₋ RFU_20BG) – (RFU₁₀₋ RFU_10BG) where: RFU₂₀ is the sample reading at 20 mins, RFU_20BG is the background sample at 20 mins, and RFU₁₀ is the sample reading at 10 mins, RFU_10BG is the background sample at 10 mins. NE activity was calculated using the following formula:
NE activity = (B/V) × D = ng/ml, where B = amount of NE from the standard curve, V = original sample volume added into the reaction well (ml), and D = sample dilution factor.

Khoury O., Clouse C., McSwain M.K., Applegate J., Kock N.D., Atala A, & Murphy S.V. (2022). Ferret acute lung injury model induced by repeated nebulized lipopolysaccharide administration. Physiological Reports, 10(20), e15400.

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Neutrophil Elastase Activity Assay

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Human neutrophils were washed and re-suspended in HBSS. Elastase release from neutrophils was evaluated using Neutrophil Elastase Activity Assay Kit (abcam). Following the 1-h incubation without or with C6 (1 μM), neutrophils (1 × 10⁶ cells) were subjected to stimulation using 10 μM LPS. Florescence was measured continually for 30 min using Fluoroskan FL. Elastase release (ng) was determined by comparing to the standard curve according to the kit manual.

Zhao R., Lopez B., Schwingshackl A, & Goldstein S.A. (2022). Protection from acute lung injury by a peptide designed to inhibit the voltage-gated proton channel. iScience, 26(1), 105901.

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Neutrophil Elastase Activity Assay

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The neutrophil elastase activity was examined using an Neutrophil Elastase Activity Assay Kit (Fluorometric, ab204730, Abcam) according to the methods described before.⁷

Zhou L., Gao R., Hong H., Li X., Yang J., Shen W., Wang Z, & Yang J. (2020). Emodin inhibiting neutrophil elastase‐induced epithelial‐mesenchymal transition through Notch1 signalling in alveolar epithelial cells. Journal of Cellular and Molecular Medicine, 24(20), 11998-12007.

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Neutrophil Activation and Enzyme Assays

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Isolated neutrophils were stimulated with 100 nM phorbol myristate acetate and seeded at a density of 25 × 10⁴ cells in a 96‐well plate in the presence or absence of 20 × 10³ stromal cells. Cells were cultured for 2 hours and 30 minutes before MPO and NE activities were assessed using the Neutrophil Myeloperoxidase Activity Assay kit (Cayman Chemical, Ann Arbor, Michigan) and Neutrophil Elastase Activity Assay Kit (Abcam, Cambridge, Massachusetts). MPO activity was assessed by incubating the cells supernatant with MPO substrate for 10 minutes. Absorbance was read using a plate reader at 650 nm. NE activity was assessed by incubating with NE substrate for 20 minutes and measuring the output on a fluorescent microplate reader at Ex/Em = 380/500 nm.

Khoury O., Atala A, & Murphy S.V. (2019). Stromal cells from perinatal and adult sources modulate the inflammatory immune response in vitro by decreasing Th1 cell proliferation and cytokine secretion. Stem Cells Translational Medicine, 9(1), 61-73.

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Neutrophil Stimulation and DNA/Elastase Assays

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Totally, 1 × 10⁶ neutrophils were stimulated using S. uberis(MOI=1, 10, and 100) or phorbol-12-myristate-13-acetate (PMA) (final concentration=100 nM)
in 200 µL RPMI 1640 medium for 6 hr at 37°C and the culture supernatants were obtained. To
evaluate the amount of DNA in the culture supernatants, a fluorescence assay was
performed, as described [8 (link)]. Culture supernatants
were mixed with SYBR green I (Toyobo, Osaka, Japan) for 30 min at 37°C. Fluorescence
intensity was measured using a 7500 L Real-Time PCR System (Applied Biosystems, Waltham,
MA, USA). Culture supernatants were assayed for elastase activity using the Neutrophil
Elastase Activity Assay Kit (Abcam), following the manufacturer’s instructions. This
experiment was conducted using seven individual bovine neutrophil samples. All assays were
performed in duplicate.

GOTO S., MIKAMI O., NAGASAWA Y, & WATANABE A. (2023). Bovine neutrophils stimulated with Streptococcus uberis induce neutrophil extracellular traps, and cause cytotoxicity and transcriptional upregulation of inflammatory cytokine genes in bovine mammary epithelial cells. The Journal of Veterinary Medical Science, 86(2), 141-149.

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