Opsonized bacteria (5 × 107/50 μL HBSS) were added to 500 μL EV (derived from 5 × 106 PMN) suspended in HBSS. During a 40 min coincubation step at 37°C, the bacterial count decreases or increases depending on the samples’ antibacterial effect and the growth of bacteria. At the end of the incubation, 2 mL ice‐cold stopping solution (1 mg/mL saponin in HBSS) was added to stop the incubation and lyse EVs. After a freezing step at −80°C for 20 min, samples were thawed to room temperature and inoculated into LB broth. Bacterial growth was followed as changes in OD using a shaking microplate reader (Labsystems iEMS Reader MF, Thermo Scientific) for 8 h, at 37°C, at 650 nm. After the end of growth phase, the initial bacterial counts were calculated indirectly using an equation similar to PCR calculation, as described previously [17 (link)].
Labsystems iems reader mf
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Labsystems iEMS Reader MF is a microplate reader designed for photometric measurements. It can be used to read absorbance in 96-well microplates. The device provides basic microplate reading capabilities.
Automatically generated - may contain errors
11 protocols using labsystems iems reader mf
1
Assessing Antimicrobial Activity of Extracellular Vesicles
2
Evaluating Immune Response of Extracellular Vesicles
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PMNs (120 μL of 2.5*107/mL) were added to 480 µl of EV samples at 37°C in a linear shaker (80 rpm) for 3 hours. Cells were centrifuged (500 g, Hermle Z216MK 45° fixed angle rotor, 10 min, 4°C) and supernatants were analyzed for IL-8, TGF-β, IL-1RA, IL-1α, IL-6, TNF-α with a human IL-8, TGF-β, IL-1RA, IL-1α, IL-6, TNF-α DuoSet sandwich ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA) in a microplate reader (Labsystems iEMS Reader MF, Thermo Scientific). We also prepared a control sample for the oZ-EV samples that contained the same amount of zymosan as the oZ-EV isolates. To achieve this, in half of the oZ-EV batch EVs were lysed whereas zymosan particles were left intact (16 (link)). We refer to this sample as “lysed oZ-EV”.
3
Extracellular Vesicle Modulation of Neutrophil IL-8 Release
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PMNs (120 μl of 2.5 × 107/ml) were added to 480 μl aEV, lysed aEV, sEV, apoEV sample or HBSS at 37°C in a linear shaker (0.18 g) for 3 h. aEV, lysed aEV, and sEV samples were prepared from 1.92 × 107 cells, whereas apoEV samples were derived from 0.96 × 107 cells.
Cells were centrifuged (500 g, 10 min, 4°C) and supernatants were analyzed for IL-8 with a human CXCL8/IL-8 DuoSet sandwich ELISA kit according to the manufacturer’s instructions (R&D Systems)38 (link) in a plate reader (Labsystems iEMS Reader MF; Thermo Scientific, Minneapolis, MN, USA).
Cells were centrifuged (500 g, 10 min, 4°C) and supernatants were analyzed for IL-8 with a human CXCL8/IL-8 DuoSet sandwich ELISA kit according to the manufacturer’s instructions (R&D Systems)38 (link) in a plate reader (Labsystems iEMS Reader MF; Thermo Scientific, Minneapolis, MN, USA).
4
Determining Pediocin PA-1 Biological Activity
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The biological activity of pediocin PA-1 was determined using a growth inhibition assay [23 (link), 105 (link)]. The sensor strains L. innocua pIMK2 and L. innocua pNZ44 were grown overnight in glass tubes (filled 20% with BHI medium) on a rotary shaker (37 °C, 230 rpm, Infors HT Multitron). The samples (culture supernatant) were stepwise diluted with BHI medium in a 96-well microtiter plate. This yielded a twofold dilution series (100 µL for each dilution). In parallel, the L. innocua cultures were diluted 1:25-fold in fresh BHI medium, and the obtained suspension was mixed 1:1 with each diluted sample. The filled microtiter plate was incubated for 6 h (37 °C, 230 rpm, Infors HT Multitron). Afterwards, the cell concentration in each well was quantified at 595 nm (Labsystems iEMS Reader MF, Thermo Fisher, Waltham, MA, USA). The pediocin PA-1 activity was then estimated from the growth inhibition data [105 (link)], using software-based parameter fitting with the sigmoidal dose response tool (Origin 2021, Northampton, UK). Throughout this study, the activity is given as bacteriocin units per mL (BU mL−1). Using the recently determined specific biological activity of purified, commercial pediocin PA-1 [23 (link)], allowed to infer peptide concentrations from biological activity measurement.
5
Antibacterial Effects of PMN-Derived Extracellular Vesicles
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Opsonized bacteria (5 × 107/50 μL HBSS) were added to 500 μL EV (derived from 5 × 106 PMN) suspended in HBSS. During a 40 min co-incubation step at 37°C the bacterial count decreases or increases depending on the samples' antibacterial effect and the growth of bacteria. At the end of the incubation, 2 mL ice-cold stopping solution (1 mg/mL saponin in HBSS) was added to stop the incubation and lyse EVs. After a freezing step at −80°C for 20 min, samples were thawed to room temperature and inoculated into LB broth. Bacterial growth was followed as changes in OD using a shaking microplate reader (Labsystems iEMS Reader MF, Thermo Scientific) for 8 h, at 37°C, at 650 nm. After the end of growth phase the initial bacterial counts were calculated indirectly using an equation similar to PCR calculation, as described previously (35 (link)).
6
Bacterial Growth Modulation by EVs
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Opsonized bacteria (5 × 107/50 μl HBSS) were added to 500 μl EV (derived from 5 × 106 PMNs) suspended in HBSS. During a 40 min coincubation step at 37°C, the bacterial count decreases or increases depending on the samples’ antibacterial effect and the growth of bacteria. At the end of the incubation, 2 ml ice-cold stopping solution (1 mg/ml saponin in HBSS) was added to stop the incubation and lyse EVs. After a freezing step at −80°C for 20 min, samples were thawed to room temperature and inoculated into LB broth. Bacterial growth was followed as changes in OD using a shaking microplate reader (Labsystems iEMS Reader MF; Thermo Scientific, Waltham, MA, USA) for 8 h, at 37°C, at 650 nm. After the end of growth phase, the initial bacterial counts were calculated indirectly using an equation similar to PCR calculation, as described previously.35 (link)
7
Extracellular Vesicle Modulation of Neutrophil IL-8 Release
8
Neutrophil Migration Assay with Extracellular Vesicles
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PMNs (120 μl of 5 × 106/ml) were added to 480 μl aEV, lysed aEV, sEV, apoEV sample or HBSS at 37°C in a linear shaker (0.18 g) for 45 min. aEV, lysed aEV, and sEV samples were prepared from 1.92 × 107 cells, whereas apoEV samples were derived from 0.96 × 107 cells. The pretreated PMN samples were placed in the wells of a 3 μm pore Corning, NY, USA transwell cell culture plate coated with 10% FBS. Every well contained 2 × 105 cells. As a chemoattractant, 100 nM N-formylmethionyl-leucyl-phenylalanine was used. After 1 h incubation at 37°C, the transwell plate was centrifuged (Eppendorf 5810 R swing-bucket plate rotor, 3,220 g, 3 min, 4°C). Transmigrated cells were counted using an acid phosphatase assay37 (link) in a plate reader (Labsystems iEMS Reader MF; Thermo Scientific).
9
Bacterial Growth Modulation by EVs
10
Neutrophil Migration Assay with Extracellular Vesicles
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