HEK-293 cells were plated on poly-d -lysine coated chambers (ibidi, Germany) in DMEM supplemented with 10% (vol/vol) FBS (DMEM/FBS). After 24 h, cells were transfected with 300 ng of rat (r) NK1R-GFP per chamber and cultured for 48 h. To identify endosomal compartments, HEK-293 cells were infected with Rab5a-GFP (resident in early endosomes) or Rab7a-GFP (late endosomes) CellLight BacMam2.0 (Thermo Fisher Scientific) for 16 h. To examine localization of nanoparticles, cells were incubated in Leibovitzś L-15 medium with DIPMA-Cy5 nanoparticles (20 μg ml−1, 30 min, 37 °C) or vehicle, followed by addition of SP (10 nM). Cells were imaged at 30 and 60 min post-SP addition using a Leica SP8 confocal microscope equipped with HCX PL APO ×40 (NA 1.30) and HCX PL APO ×63 (NA 1.40) oil objectives. Images were analysed using Fiji36 (link) and deconvolved with Huygens Professional version 18.04 (Scientific Volume Imaging, http://svi.nl ), using the CMLE algorithm with a signal-to-noise ratio of 10 and 100 iterations. Co-localization was evaluated by determination of the Manders overlap coefficient25 .
Hcx pl apo 40
Manufactured by Leica
Sourced in Germany
The HCX PL APO ×40 is a high-quality objective lens designed for microscopy applications. It features a long working distance and high numerical aperture, providing excellent optical performance and resolution.
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Lab products found in correlation
Dm6000b microscope, by Leica (3 mentions)
Tcs sp5 aobs, by Leica (2 mentions)
Lsm 510 meta, by Zeiss (2 mentions)
Celllight bacmam 2, by Thermo Fisher Scientific (1 mentions)
Huygens professional version 18, by Scientific Volume Imaging (1 mentions)
Sp8 confocal microscope, by Leica (1 mentions)
N bk7 600 grit, by Thorlabs (1 mentions)
Wph05m 1064, by Thorlabs (1 mentions)
Pbs cm1 pbs253, by Thorlabs (1 mentions)
Fgs900s, by Thorlabs (1 mentions)
Prolong mounting media, by Thermo Fisher Scientific (1 mentions)
Las x 3d version 3, by Leica (1 mentions)
Tcs sp5, by Leica (1 mentions)
10 protocols using hcx pl apo 40
1
Nanoparticle Trafficking in Transfected HEK-293 Cells
2
Digestion of NETs in Paraffin Sections
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The digestion of NETs in paraffin section with external DNase was conducted as described previously (15 (link)). Briefly, the digestion was conducted in one slide for 30 minutes at 37°C. A serial cut of the paraffin section was handled and stained in parallel without adding DNase. Samples were recorded using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with an HCX PL APO ×40 0.75–1.25 oil immersion objective The settings were adjusted using isotype control antibodies in separate preparations. Stitched images were combined using Adobe Photoshop CS6.
3
Bright-field Microscopy with Heating Beam
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Imaging was done using bright-field on a home-built microscope coupled to a laser source. A commercial light-emitting diode (λ = 505 nm THORLABS) with a diffuser (ground glass N-BK7 600 grit, THORLABS), a condenser and an iris, to achieve Köhler illumination. The scattered light was picked up by the microscope objective (HCX PL APO ×40 NA = 0.85, LEICA), and a tube lens (B&H), and detected by a digital camera (DCC1545M, IMAGING SOURCE) and acquired using commercial video recording software (IC CAPTURE, IMAGING SOURCE). A heating beam was introduced on a separate optical path (see Supplementary Information ). A 1064 nm laser beam (YLR-10-1064-LP, IPG Photonics) was passed through a zero-order half-plate (WPH05M-1064 Thorlabs) and contracted using a custom Galilean telescope to achieve a ~300 μm beam (see Supplementary Information ). The laser beam was introduced into the sample using a polarizing beam splitter (PBS CM1-PBS253 THORLABS) and its intensity at the sample was controlled by a combination of the electronic laser head controller and by adjusting the half-plate, and was measured using an optical power meter (PM100D power meter, with S175C sensor, THORLABS). In order to eliminate laser intensity before the camera, stained glasses (FGS900S, THORLABS) were stacked after the objective.
4
Confocal Microscopy Imaging of Labeled Specimens
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Whole-mount specimens were viewed and scanned with an LSM 510 Meta (Carl Zeiss, Jena, Germany) or with a Leica TCS SP2 or SP5 (Leica, Bensheim, Germany) laser-scanning confocal microscope system equipped with Plan-Neofluar 40× (Zeiss), HCX PL APO 40× (Leica), PL APO 63× (Zeiss, Leica) oil immersion objectives or a PL APO 63× glycerol objective (Leica). All fluorescent labels (Alexa 488, Exmax 490 nm, Emmax 508 nm; Cy3, Exmax 550 nm, Emmax 570 nm; Cy5, Exmax 650 nm, Emmax 674 nm) were excited using an argon laser at 488 nm, an He/Ne 1 laser at 543 nm, and an He/Ne laser at 633 nm. Alexa 546 (phalloidin) stainings were excited with an He/Ne 1 laser at 543 nm. Immunostained specimens were scanned at a resolution of 1024 × 1024 pixels with a pinhole size of 1 airy and a distance between optical sections of 1 μm. Final images resulted either from maximal projections of a certain number of optical sections or from single representative sections out of a stack of optical sections. Reconstructions were obtained using modified camera lucida techniques on the projection of a series of high-resolution tagged image files obtained from Z-stack conversion. The final reconstructed image was scanned using an HP Pro scanner (Corvallis, OR) and processed in Adobe Photoshop (San Jose, CA). All image plates were aligned and adjusted for contrast with Adobe Photoshop.
5
DNA Fiber Analysis of Replication
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Exponentially growing MEFs or single-cell suspensions of fetal livers were pulse-labeled with 50 μM CldU (20 min) followed by 250 μM IdU (20 min). Labeled cells were harvested and resuspended in 0.2 M Tris pH 7.4, 50 mM EDTA and 0.5% SDS. Stretched DNA fibers were prepared as described52 (link). For immunodetection of labeled tracks, fibers were incubated with primary antibodies for 1 h at RT and the corresponding secondary antibodies for 30 min at RT, in a humidity chamber. DNA was stained with anti-ssDNA to assess fiber integrity. Fiber images were obtained in a DM6000 B Leica microscope with an HCX PL APO 40 ×, 0.75 NA objective. The conversion factor used was 1 μm=2.59 kb. In each assay, >300 individual tracks were measured for FR estimation, >100 fibers containing two or more origins were analyzed for IOD estimation, >200 labeled fibers were counted for fiber length and origin density, and >180 bidirectional forks were counted for fork symmetry.
6
DNA Replication Dynamics Analysis
7
DNA Fiber Analysis of Replication Dynamics
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Cells were pulse-labelled sequentially with 50 μM CldU (20 min) and 250 μM IdU (20 min), harvested and resuspended in PBS (0.5 × 106 cell/ml). 2 μl drops of cell suspension were placed on microscope slides and lysed with 0.5% SDS, 0.2 M Tris pH 7.4, 50 μM EDTA in 10 μl for 6 min at RT. Slides were tilted 15°C to spread DNA fibers, air-dried, fixed in –20°C methanol:acetic acid (3:1) for 2 min and stored at 4°C overnight. Slides were then incubated in 2.5 M HCl (30 min/RT) to denature DNA and washed (3×) in PBS. Blocking solution (1% bovine serum albumin in PBS, 0.1% Triton X-100) was added for 1 h at RT. Slides were incubated with anti-CldU, IdU and ssDNA primary antibodies for 1 h at RT, washed and incubated with the corresponding secondary antibodies for 30 min. Prolong mounting media (Invitrogen) was used. Images were acquired in a DM6000 B Leica microscope with an HCX PL APO 40×, 0.75 NA objective. Fork rate values were derived from the length of IdU tracks, measured using ImageJ software, and a conversion factor of 1 μm = 2.59 kb (36 (link)). >300 tracks were measured per condition. The percentage of origins activated during the first pulse (green-red-green tracks) was quantified relative to all replicative structures containing red signal. >500 total structures were scored in each case. Three biological replicates of each experiment were performed.
8
Quantifying Neutrophil Extracellular Traps
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Samples were recorded using a Leica TCS SP5 confocal inverted-base fluorescence microscope with a HCX PL APO 40 × 0.75–1.25 oil immersion objective. Settings were adjusted with control preparations using an isotype control antibody. For each sample 6–7 randomly selected images per independent experiment were acquired and used for quantification of the granulocyte number and the induced ET forming cells with an occurrence of a distinct extracellular off-shoot of the cells which is positive for histone-DNA complexes. For the 3D picture and movie, z-stacks were collected and analyzed with LAS X 3D Version 3.1.0 software from Leica. Fifty-six z-stack pictures were used and the background set to gray by standard software settings. The movie was made with the movie maker in the software.
9
Quantification of Neutrophil Extracellular Traps
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The NETs were quantified as previously described [71 (link)]. Six randomly chosen pictures were taken, using a Leica TCS SP5 AOBS confocal inverted-base fluorescence microscope with an HCX PL APO 40 × 0.75–1.25 oil immersion objective. The pictures were taken at predefined positions on two slides (see Figure A4 , Appendix A ). Only if an artefact (e.g., air bubble) was present was another adjacent area selected. The focus was set on the nuclei (blue channel). The cells were counted manually, using ImageJ software (version 1.52q; National Institute of Health, Bethesda, MD, USA) with the Cell counter plugin. All of the NET negative and positive neutrophils were counted. The neutrophils on the border of an image (not fully visible) were not counted at all. In each sample, a minimum of 300 cells were counted. A neutrophil was counted as positive if an evident off-shoot of DNA was visible, or if at least two of the following criteria were found: enlarged nucleus; decondensed nucleus; or blurry rim. The percentage of the NET-positive neutrophils was calculated (NET-activated cells). An average from the counted NET-activated cells from the six pictures from each sample was calculated.
10
Immunofluorescence of Matrigel-cultured Cysts
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Cysts seeded on Matrigel into µ-Slide 8 well ibiTreat chambers were fixed at different time point after seeding in PBS 2% PFA for 20 min, washed 3 times in PBS 1% Glycine (quenching) and left 2 days in PBS 1x at 4 °C. Cysts were permeabilized in PBS + 0.5% Triton X-100 and quickly washed in IF-buffer (PBS, 0.2% Triton X-100, 0.1% BSA, 0.05% Tween). Samples were then incubated in blocking solution (IF-buffer + 5% BSA) for 1 h and 30 min. After 1 wash in IF-buffer, cysts were incubated with primary antibodies in IF-buffer, overnight at 4 °C. Samples were washed 3 times in IF-buffer (5 min each) and incubated with the appropriate secondary antibody and/or phalloidin for 1 h and 30 min in the dark. After 1 wash in IF-buffer (5 min) and 2 washes in PBS samples were incubated with Dapi (5 min). Samples were washed 2 times in PBS and them left in PBS for confocal analysis. Images were acquired with confocal microscopes of the Leica SP series (see below for the detailed information) equipped with a HCX PL APO 40×/0.75–1.25 oil immersion objective.
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