br Quantification of NETs br NETs sca
2.5. Quantification of NETs
NETs scaﬀolds in coculture were digested with 250 mU/mL of mi-crococcal nuclease (LS004797, Worthington Biochemical Corp.) and then stained with 2.5 μM Sytox Orange (S34861, Molecular Probes) for 10 min at room temperature. Quantification was measured using a fluorometer (Synergy H1 Hybrid Reader, BioTek) every min for up to 300 min.
2.6. Preparation of cell-free NETs and quantification of DNA and protein
Neutrophils (1 × 105/mL) were seeded in 6-well culture plates with 2 mL of phenol red–free RPMI-1640 (11835030, Invitrogen). Following stimulation with BCG (MOI = 10) for 4 h, the medium was removed and Sevoflurane were gently washed. After addition of 1 mL RPMI (phenol red–free) to the adherent film and vigorous agitation followed by cen-trifugation at 220 ×g for 5 min, the supernatant was collected, as re-ported . DNA and protein were quantified using Picogreen dsDNA kit (P11495, Invitrogen) and Micro-BCA protein assay kit (PI23235, Pierce Biotechnology) respectively, according to the instructions. Ad-ditional micrococcal nuclease digestion and centrifugation at 2200 ×g for 10 min were required for obtaining cell-free NETs. Less than 3 cells/ HPF and a BCG count < 5 CFU/mL, as detected by microscopy and plate culture, respectively, was necessary to exclude residual eﬀects in the following tests.
2.7. Co-culture of BCG-stimulated cancer cells with neutrophils
The human urinary BC lines, T24 (ATCC HTB-4) and 5637 (ATCC HTB9), were adopted. Cells were cultured in cc RPMI-1640 (21875091, Invitrogen) supplemented with 10% FCS at 37 °C in a humidified at-mosphere and 5% CO2. T24 and 5637 cells at 70% -80% confluence were stimulated with BCG (MOI = 10) for 6 h. The supernatants were collected by centrifugation at 6000 ×g for 10 min and used for the stimulation experiments and cytokine detection. Neutrophils (1 × 105 cells/mL) were incubated for 3 h, in 1 mL of the prepared supernatants or fresh medium. Intact neutrophils (1 × 105 cells/mL) incubated with BCG-treated or untreated cancer cells (1 × 104 cells/ mL) were washed repeatedly to remove all traces of agents and NETs formation was observed.
2.8. Measurement of viability, cell cycle, apoptosis, migration and invasion of tumor cells
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seeded at a density of 1 × 105 cells/mL to reach 90% confluency, were treated with diﬀerent amounts of NETs (0.5, 1, and 2 ×), and the su-pernatant with no NETs served as a negative control. The morphologies were noted and the following tests were performed.
Fig. 2. NETs formation was induced by BCG-activated tumor cells.
A, as shown by CLSM after DAPI staining, NETs formation was abundant in the neutrophils co-cultured with tumor cells pre-activated by BCG, but limited in the ones co-cultured with non-stimulated tumor cells (red arrows indicated tumor cells). B, representative images showed that the supernatants of BCG-activated tumor cells induced NETs that contain chromatin and granule proteins, as confirmed by extracellular multi-fluorescence co-localization of DNA, cit-H3, and NE. C, the super-natants of BCG-activated tumor cells induced neutrophil morphologic changes to liberation of fibrous material, that was fine and sparse, resembling the material induced by PMA (reference to Fig. 1B). D, the supernatants of BCG-activated tumor cells induced more NETs than the non-activated ones did. E, the levels of IL-8, TNF-α and HMGB1 were determined with ELISA, and shown to be markedly increased in the supernatants of tumor cells stimulated by BCG compared with those in the non-stimulated supernatants. F, administration of anti-IL-8 and anti-TNF-α antibodies in the supernatants of BCG-activated tumor cells significantly reduced NETs formation, with non-stimulated neutrophils served as the control. *P < .05, **P < .01. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
After NETs treatment, 100 μL of CCK-8 (CK04, Dojindo) was added to the cell suspension with media containing only NETs as the blank. Following incubation, absorbance at 450 nm was determined using a microplate reader (Multiskan EX, Thermo, USA). The growth inhibition rate (%) was calculated as follows: (1 − Abstest/Abscon) × 100%.
Cancer cells were treated with NETs, 1% Triton X-100 (high control) or untreated (low control). LDH release was assessed using a cytotoxi-city detection kit (11,644,793,001, Roche Applied Science) according to the manufacturer's instructions and represented as the average ab-sorbance at 490 nm. Cytotoxicity (%) was calculated as follows: (NETs −low control)/(high control −low control) × 100%.