Trapalyzer: A computer program for quantitative analyses in fluorescent live-imaging studies of Neutrophil Extracellular Trap formation.

This data set contains a set of fluorescent microscopy images of a co-culture of neutrophil cells and E. coli bacteria used to study the Neutrophil Extracellular Trap (NET) formation stimulated by bacteria. 

NETs and live cells were visualized with a double fluorescent staining of DNA using Hoechst 33342 and SYTOX Green. 

Reagents.

Roswell Park Memorial Institute (RPMI) 1640 medium, HEPES, SYTOX^TM Green, and Hoechst 33342 were purchased from Thermo Fisher Scientific (Waltham, USA). LB broth was purchased from Sigma Aldrich (St Louis, MO, USA).

Preparation of blood neutrophils.

Neutrophils were obtained from peripheral blood of one healthy blood donor. Blood sample was purchased at Local Blood Donation Centre and according to local regulations, the blood donor enabled blood donation center to sell their blood samples for scientific purposes and the consent of bioethical committee was not required. Blood was collected into a citrate tube and processed within 2 hours from collection. Neutrophils were isolated using density gradient centrifugation followed by polyvinyl alcohol sedimentation, exactly as described in [1]. Isolated neutrophils were suspended in RPMI 1640 medium with 10 mM HEPES (RH).  

Preparation of bacteria.

Escherichia coli (American Type Culture Collection(ATCC) 25922 strain) were grown overnight in LB broth with shaking. In the morning, an aliquot of bacterial culture was taken, diluted 100 x in a fresh LB medium and grown for subsequent 2-3 hours. Subsequently, bacterial cultures were washed and resuspended in RH medium.

Co-culture of neutrophils with bacteria
Neutrophils were seeded into the wells of 48-well plates at the density of 2 ⨉ 10⁴ cells/well and allowed to settle for 30 minutes at 37°C, 5% CO2. Subsequently, E. coli was added into the appropriate wells at the multiplicity of infection of 4 or 1 (E.coli: neutrophil). Neutrophils incubated without bacteria were used as a control group. A technical duplicate for each condition was prepared.  
For each intended timepoint (t=0, 60, 90, 120, 180 minutes), a separate 48 well plate was prepared. The plates were centrifuged for 5 minutes at 250 g to allow the contact of bacteria with neutrophils. The plates were incubated at 37°C, 5\% CO2 for a specified time and then the samples were stained with SYTOX^TM Green (100 nM) and Hoechst 33342 (1.25 μM) for 10 minutes. Four images of each well were taken with Leica DMi8 fluorescent microscope equipped with a 10× magnification objective (Leica, Wetzlar, Germany). Overall, 120 images have been obtained.

2019_04_24--ecoli_neu_tiff_channel_merged.zip: Images in .tif format, each containing 5 channels: channel 1 for SYTOX Green fluorescent stain (green fluorescence), channel 2 for Hoechst 33342 fluorescent stain (blue fluorescence), and three channels for transmission light encoded in RGB values. 

2019_04_24--ecoli_neu_tiff_raw_exported.zip: Images split by different light sources: transmission light (_ch00.tif), SYTOX Green fluorescence (_ch01.tif), Hoechst 33342 fluorescence (_ch02.tif).

[1] Bystrzycka W, Moskalik A, Sieczkowska S, Manda-Handzlik A, Demkow U, Ciepiela O. The effect of clindamycin and amoxicillin on neutrophil extracellular trap (NET) release. Cent Eur J Immunol. 2016;41(1):1-5. doi:10.5114/ceji.2016.58811