Bull serum albumin bsa

Sulfamide and m-phenylenediamine (m-PD) were obtained from J&K Scientific Ltd. (Beijing, China). Sodium pyrophosphate and amino acids were obtained from Sangon Biotechnology Co., Ltd. (Shanghai, China). Alkaline phosphatase (ALP, EC 3.1.3.1, 2 kU) was purchased from Sigma (USA). Glucose oxidase (GOX), thrombin, trypsin, pepsin, lysozyme, cysteine and bull serum albumin (BSA) were purchased from Solarbio (Beijing, China). Other reagents were purchased from Tianjing Chemical Reagents Company (Tianjing, China). All chemicals were used as received without further purification. HEPES buffer (10 mM, pH 7.4) was used throughout the experiments. Ultrapure water was obtained from a Milli-Q Plus system (Millipore, Bedford, MA, USA) and used in all experiments.

Neutrophil suspensions (0.5 ml) were seeded at a density of 4×10⁵ cells/ml in 24-well plates on poly-L-lysine coated coverslips. These suspensions were seeded in either 0.2 ml yeast cell suspensions at a final density of 2×10⁵ cells/ml or 0.2 ml phorbol myristate acetate (PMA; MCE, Monmouth Junction, NJ, USA) at a final concentration of 25nM or in 0.2 ml RPMI 1640 medium. One 24-well plate was then placed in a water bath at a temperature of 41℃, while a second 24-well plate was placed in the water bath at a temperature of 37℃. After 30 minutes, two 24-well plates were placed in a cell incubator at 37℃ in 5% CO₂ atmosphere for 4 hours. The cells were then fixed and permeabilized, followed by blocking with 1% bull serum albumin (BSA; Solarbio, Beijing, China). Next, cells were incubated with anti-human neutrophil elastase antibody (R&D Systems, Minneapolis, MN, USA), and detected with Alexa Fluor 488 goat anti-mouse (CST, Boston, MA, USA), followed by staining with DAPI. Imaging data were acquired using a Live Cell Station (Bio-Tek, Winooski, VT, USA). The number of NETs and neutrophils were counted and the percentage of NETs formed was calculated. The experiment was replicated five times.