Ixonem 897e back illuminated emccd camera

Images were captured using a Nikon TE-2000U inverted microscope (Nikon, Melville, NY) coupled to an iXonEM+ 897E back illuminated EMCCD camera (Andor, Belfast, U.K.) outfitted with a Bioptechs (Butler, PA) stage heater and 20x, Nikon Plan Apochromat objective. Bright field images were captured using Elements software (Nikon). For fluorescence microscopy, a xenon lamp illuminated cells through a 33-mm ND4 filter and 20x Nikon Plan Apochromat objective using a Nikon B2-A long pass emission filter set cube.

Cells collected from peritoneal lavage were re suspended (1:20) in 1× PBS and placed on tissue culture plates coated with 1% BSA and incubated at 37°C for 1–2 hrs. 5µM of Sytox® green (Thermo Fischer Sci, Waltham, MA,) was added 10 min prior to imaging. Images were captured using a Nikon TE-2000U inverted microscope (Nikon, Melville, NY) coupled to an iXon^EM + 897E back illuminated EMCCD camera (Andor, Belfast, UK). Bright field images were captured using NIS-Elements software (Nikon). A xenon lamp illuminated cells through a 33 mm ND4 filter and 20× Nikon Plan Apochromat objective using a Nikon B2-A long-pass emission filter set cube.
For flow cytometry, all the antibodies used for analysis were purchased from eBioscience (San Diego, CA). LY6G⁺ cell populations in the peritoneal lavage were determined with FACS Array flow cytometer (BD Bioscience, San Jose, CA) and analyzed with FlowJo software (Tree Star, Ashland, OR) was used for analysis [31 (link)].

Thioglycolate elicited neutrophils were isolated and cultured on
Poly-L-lysine coated plates for 4 hours at 37°C post 20nM PMA
stimulation to induce NET production. 5μM of Sytox®
green (Thermo Fischer Sci, ‎Waltham, MA,) was added 10 minutes
prior to imaging. Images were captured using a Nikon TE-2000U inverted
microscope (Nikon, Melville, NY) coupled to an iXon^EM+ 897E back illuminated EMCCD camera (Andor, Belfast, UK).
Bright field images were captured using NIS-Elements software (Nikon). A
xenon lamp illuminated cells through a 33 mm ND4 filter and 20×
Nikon Plan Apochromat objective using a Nikon B2-A long-pass emission
filter set cube.