NET formation was quantified by histone area. All images were obtained by using Leica confocal microscope (TCS-SP5-MP-SMD) and processed under Leica LAS AF software. Histone image of neutrophil were analyzed using MetaMorph® software. Briefly, NETs were visualized in at least five random fields (40 × magnification), and signal intensity of histone per fields was individually measured; and the pixels of each image were converted into area (μm2) using a calibration unit (0.8333). Mean of histone area was from the five independent fields (Supplementary Fig. 24 ) were denoted as NET histone area (μm2).
Tcs sp5 mp smd
Manufactured by Leica
The TCS-SP5-MP-SMD is a laser scanning confocal microscope system designed for multi-photon imaging. It features a spectral detection system and supports simultaneous detection of multiple fluorescent signals.
Automatically generated - may contain errors
Lab products found in correlation
Las af software, by Leica (1 mentions)
Permeabilization buffer, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 conjugated donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 546 conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Dapi fluoromount g mounting medium, by Southern Biotech (1 mentions)
4 protocols using tcs sp5 mp smd
1
Quantifying Neutrophil Extracellular Traps
2
Immunofluorescence Staining of Mammospheres
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Cells were fixed in 4% paraformaldehyde at room temperature for 10 minutes. Primary antibodies were used at the dilutions suggested by the manufacturer. Cells were permeabilized with a permeabilization buffer (eBioscience) before staining with ALDH1, collagen type II, nestin, βIII-tubulin and GFAP. Secondary antibodies (1:100) labeled with Alisa488, Alisa594, PE or APC were added and incubated for one hour at room temperature. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). Stained mammospheres and monolayer cultured cells were imaged on a Confocal Microscope and Single Molecule detection system (Leica, TCS-SP5-MP-SMD).
3
Immunofluorescence Staining of Macrophages
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Murine and human macrophages were fixed and permeabilized, followed by incubation with anti-CLEC18 (60 μg/ml), anti-TLR3 (20 μg/ml), or anti-TLR7 (20 μg/ml) Abs at 4 °C for 16–18 h, followed by incubation with Alexa Fluor 488-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 715-545-150) or Alexa Fluor 546-conjugated goat anti-rabbit IgG (Life Technologies, #A-11035). Samples were mounted with coverslips and observed under a confocal microscope (TCS-SP5-MP-SMD, Leica) and analyzed by the SymPhoTime software.
4
Visualization of circRNA in HeLa cells
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HeLa cells were grown in 24-well overnight, and then fixed in 4% paraformaldehyde solution at room temperature for 10 min, rinsed with phosphate-buffered saline twice. Cells were incubated in hybridization buffer (2× Magnetic Particle Concentrator (SSC) , 10% dextran sulfate, 10% deionized formamide in nucleus free water) with Cy5-labeled probes antisense to the back-splice junction of target circRNAs at 40°C for 16 h. After hybridization, cells were washed with 2× SSC at 37°C for 30 min. Slides were mounted onto glass slides using DAPI-Fluoromount-G mounting medium (SouthernBiotech). The fluorescence signals were detected by confocal microscope system (Leica TCS-SP5-MP-SMD), and the signal intensities were quantified by the ImageJ software.
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