Trypanosomes in the logarithmic phase of growth were washed in BME at 4 °C and adhered to glass coverslips coated with 0.1 % of poly-l -lysine in PBS (pH 7.2), for 20 min. Then, cells were washed, fixed in methanol at 20 °C, and incubated at 22 °C with 1 μg/mL Hoechst H33342 (Molecular Probes) in BME, for 15 min. Coverslips were mounted using 0.2 M N-propyl-gallate (Sigma, USA) in glycerol and 0.01 M PBS (pH 7.2), and analyzed by epifluorescence microscopy using a Zeiss Axioplan II light microscope equipped with a Color View XS digital camera.
Hoechst h33342
Manufactured by Thermo Fisher Scientific
Hoechst H33342 is a fluorescent dye that binds to the minor groove of DNA. It is commonly used in biological research applications to stain and visualize nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Harmony 4.9 high content imaging and analysis software, by PerkinElmer (3 mentions)
Opera phenix microscope, by PerkinElmer (3 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (2 mentions)
N propyl gallate, by Merck Group (1 mentions)
Axiovision 4, by Zeiss (1 mentions)
Axiocam ccd camera, by Zeiss (1 mentions)
Axio observer z1 microscope, by Zeiss (1 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (1 mentions)
Water immersion lens, by PerkinElmer (1 mentions)
Image it tmrm reagent, by Thermo Fisher Scientific (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Euromedex (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Texas red phalloidin, by Thermo Fisher Scientific (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Oligomycin, by Enzo Life Sciences (1 mentions)
Annexin 5 alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 antibody, by Thermo Fisher Scientific (1 mentions)
Xpress tag, by Thermo Fisher Scientific (1 mentions)
10 protocols using hoechst h33342
1
Visualizing Trypanosomes via Epifluorescence
2
Quantifying Cell Detachment under Fluid Shear
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In order to quantify the number of cells detached as a function of time and shear stress under fluid shear, we counted labelled nuclei. The cell nuclei were labelled using Hoechst H33342 (Molecular Probes) at 1:1000 ratio of the 10 mg/ml slock solution. The fluorescence imaging was done using a motorized Zeiss AxioObserver-Z1 microscope, an Axiocam CCD camera, and the AxioVision 4.8 software, all supplied by Zeiss. The counting of labelled nuclei was done using in-house MATLAB code that involved counting of bright objects of a specified size range. Another in-house code was written in MATLAB to measure cell areas. Both the codes were written using the in-built Matlab function "regionprop".
3
Evaluating Mitochondrial Activity and Viability
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The MTT colorimetric assay was used to measure the effects of 2,4-D and 2,4,5-T treatments on mitochondrial function [35, 36 (link)]. Absorbances were measured at 540 nm in a SpectraMax M5 Microplate Reader (Molecular Dynamics, Sunnyvale, CA, USA). After discarding the supernatants and rinsing the wells with phosphate-buffered saline (PBS), the cells were stained with Hoechst H33342 (10μg/ml in PBS) (Thermo Fisher Scientific, Cambridge, MA) for 15 min protected from ambient light with aluminum foil. Fluorescence (Ex360 nm/Em460 nm) intensity corresponding to nuclear staining was measured in a SpectraMax M5 Microplate Reader. Although H33342 labels live and dead cells, the PBS rinses dislodge and remove unfixed non-viable cells, preventing their inclusion in this component of the assay. Using this approach, H33342 fluorescence intensity increases linearly with viable cell culture density in 96-well plates under non-saturated conditions [37 (link)]. The calculated MTT/H33342 ratios were used as indices of relative mitochondrial activity/cell viability. These assays were performed in replicates of 16 and repeated twice. and Hoechst H33342 fluorescence was used as an index of cell number/viability.
4
High-Content Imaging of Mycobacterial Infection
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30,000 iPSDM were seeded into a ViewPlate glass bottom 96- well plate and treated with LLOMe, Silica or infected with Mycobacterium tuberculosis as described above. The plate was sealed with parafilm and placed in a pre-heated (37 °C) Opera Phenix microscope with 40× or 60× water-immersion lens (PerkinElmer) with 5% CO2.
Capture settings were: Image-iT TMRM Reagent (I34361, Thermo Fischer) and MitoTracker Red CMXRos (M7512, Thermo Fischer) were excited with the 561 nm laser at 10% power with 100 ms exposure. MitoTracker Deep Red FM (M22426, Thermo Fischer), iABP probe and Mtb E2crimson were excited with the 640 nm laser at 10% power with 100 ms exposure. The mitoTimer construct was excited with the 488 nm laser and the 561 nm laser at 10% power and 100 ms exposure. The pHyPer-dMito construct was excited with the 405 and 488 nm lasers and emission was collected at 510 nm for both excitations. At least 20 fields per well were imaged in all the experiments. Images were acquired at 1020 × 1020 pixels using Harmony 4.9 high content imaging and analysis software (PerkinElmer). Cystatin B c-GFPSpark-tag, RAB7-GFP, RAB7(Q67L)-GFP and Lamp1-mNeonGreen- expressing cells were excited using the 488 nm laser at 10% power with 50 ms exposure. Hoechst H33342 (H3570, Thermo Fischer) was excited using the 405 nm laser at 15% power with 100 ms exposure.
Capture settings were: Image-iT TMRM Reagent (I34361, Thermo Fischer) and MitoTracker Red CMXRos (M7512, Thermo Fischer) were excited with the 561 nm laser at 10% power with 100 ms exposure. MitoTracker Deep Red FM (M22426, Thermo Fischer), iABP probe and Mtb E2crimson were excited with the 640 nm laser at 10% power with 100 ms exposure. The mitoTimer construct was excited with the 488 nm laser and the 561 nm laser at 10% power and 100 ms exposure. The pHyPer-dMito construct was excited with the 405 and 488 nm lasers and emission was collected at 510 nm for both excitations. At least 20 fields per well were imaged in all the experiments. Images were acquired at 1020 × 1020 pixels using Harmony 4.9 high content imaging and analysis software (PerkinElmer). Cystatin B c-GFPSpark-tag, RAB7-GFP, RAB7(Q67L)-GFP and Lamp1-mNeonGreen- expressing cells were excited using the 488 nm laser at 10% power with 50 ms exposure. Hoechst H33342 (H3570, Thermo Fischer) was excited using the 405 nm laser at 15% power with 100 ms exposure.
5
Immunofluorescence Staining of Cells
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Cells were washed in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde (PFA) (Euromedex) for 10 min at room temperature (RT). For ion channel staining, cells were fixed for 10 min with PFA 1% (RT) and 10 min in methanol at − 20 °C. Cells were then incubated in a blocking solution (PBS supplemented with 1% bovine serum albumin (BSA), 2% normal goat serum (NGS), 0.1% Triton X-00 for 1 h (RT). Primary antibodies were added, and cells were incubated either for 2 h at RT or overnight at 4 °C. Secondary antibodies were added for 1 h at RT in PBS supplemented with BSA and NGS. Antibodies are listed in Supplementary Tables 2 and 3. Nuclei were stained with Hoechst H33342 (Thermo Fisher Scientific). Slides were mounted with Fluoromount G (Southern Biotech).
6
High-Content Live-Cell Imaging of Mtb Infection
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High-content live-cell imaging: 50,000 iPSDM were seeded into a 96-well glass bottom Viewplate (6005430; Perkin Elmer) or olefin-bottomed 96-well plate (6055302; Perkin Elmer) and infected with Mtb as described above. The plate was sealed with parafilm and placed in a preheated (37°C) Opera Phenix microscope with either a 40× 1.1NA or 63× 1.15NA water immersion objective (Perkin Elmer) with 5% CO2. Images were acquired in confocal mode, a binning of 1. Capture settings were: Mtb E2crimson was excited with the 640 nm laser at 10% power with 100 ms exposure. The pHyPer-cyto, pHyPer-pexo, and pHyPer-endo construct were excited with the 405 and 488 nm lasers, and emission was collected at 510 nm for both excitations. At least 20 fields per well were imaged in all the experiments. Images were acquired at 1,020 × 1,020 pixels using Harmony 4.9 high content imaging and analysis software (PerkinElmer). Hoechst H33342 (H3570; Thermo Fisher Scientific) was excited using the 405 nm laser at 15% power with 100 ms exposure. Fluorescence was detected using a 16-bit scMOS camera.
7
High-Content Live-Cell Imaging of Mtb Infection
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High-content live-cell imaging: 50,000 iPSDM were seeded into a 96-well glass bottom Viewplate (6005430; Perkin Elmer) or olefin-bottomed 96-well plate (6055302; Perkin Elmer) and infected with Mtb as described above. The plate was sealed with parafilm and placed in a preheated (37°C) Opera Phenix microscope with either a 40× 1.1NA or 63× 1.15NA water immersion objective (Perkin Elmer) with 5% CO2. Images were acquired in confocal mode, a binning of 1. Capture settings were: Mtb E2crimson was excited with the 640 nm laser at 10% power with 100 ms exposure. The pHyPer-cyto, pHyPer-pexo, and pHyPer-endo construct were excited with the 405 and 488 nm lasers, and emission was collected at 510 nm for both excitations. At least 20 fields per well were imaged in all the experiments. Images were acquired at 1,020 × 1,020 pixels using Harmony 4.9 high content imaging and analysis software (PerkinElmer). Hoechst H33342 (H3570; Thermo Fisher Scientific) was excited using the 405 nm laser at 15% power with 100 ms exposure. Fluorescence was detected using a 16-bit scMOS camera.
8
Visualizing Endothelial Cell Junctions
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To analyze the area of opening of interendothelial junctions (OIJ), the actin filaments of PMLEC were marked. In order to achieve that, the lung endothelial cells were plated in 24 well plates (7 × 104 cells/well), adhered to gelatin on glass coverslips, and maintained at 37°C and 5% CO2. The cells were stimulated with PbA lysate for 3 h, after incubation with hemin (5, 10, and 20 μM during 24 h), or solely with DMEM culture medium, supplemented with 20% FBS, in triplicate. Subsequently, the cells were fixed with 3.7% formaldehyde, permeabilized with acetone at −20°C, and blocked with bovine serum albumin solution (1% BSA). Actin was marked with Texas Red Phalloidin (Life Technologies) by 20 minutes. The cell nuclei were marked with Hoechst (H33342, Life Technologies).
Each slide, with fully confluent cells, was chosen randomly and ten to twenty pictures were taken and scanned in a “zig-zag” way, from top to bottom. The images were acquired in the fluorescence Axio Imager M2 (Zeiss) microscope using the Axio Cam HRc (Zeiss) and the software Axio Vision, version 4.9.1.0. The total area of OIJ was measured in each picture using the software Gimp, version 2.8.16 (https://www.gimp.org/ ).
Each slide, with fully confluent cells, was chosen randomly and ten to twenty pictures were taken and scanned in a “zig-zag” way, from top to bottom. The images were acquired in the fluorescence Axio Imager M2 (Zeiss) microscope using the Axio Cam HRc (Zeiss) and the software Axio Vision, version 4.9.1.0. The total area of OIJ was measured in each picture using the software Gimp, version 2.8.16 (
9
Multiparametric Cell Imaging Protocol
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Cell imaging was performed in 384-well plates. For living cells, 30 min prior imaging, 25 μl of culture medium were removed and replaced by 25 μl of 2× mix of dyes, to a final concentration of 200 ng/ml of Hoechst H33342 (nuclear staining; Life Technologies), 10 nM of TMRM (mitochondrial membrane potential; Life Technologies), and/or 1/100 Annexin-V-Alexa Fluor 647 (early apoptosis; Life Technologies). If chemical inhibitors of the ETC were used in the experiments, they were added to hMDMs at the indicated times points at the following concentrations: 5 μM oligomycin (Enzo), 100 μM DCCD (Sigma), 10 μM FCCP (Tocris), and 50 μM BTB06584 (Sigma). Once the assay was performed, cells were fixed with 4% PFA, permeabilized with 0.1% Triton-X100, blocked with 1% BSA, and stained with primary mouse antibodies against Xpress tag (1:100, Invitrogen) and secondary anti-mouse Alexa Fluor 488 antibodies (1:1000, Invitrogen). Image acquisitions of multiple fields (9–25) per well were performed on an automated confocal microscope (Opera Phenix, PerkinElmer) using ×40 or ×60 objective, excitation lasers at 405, 488, 561, and 640 nm, and emission filters at 450, 540, 600, and 690 nm, respectively.
10
Mitochondrial and Lipid Dynamics in ILC2s
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Mitochondrial mass, membrane potential and FA uptake of freshly sorted or cytokine-activated ILC2s were assessed by staining cells with 50 nM MitoTracker Green FM (Thermo Fisher), 25 nM TMRM (Sigma-Aldrich) and BODIPY FL-C16 (Thermo Fisher), respectively, for 30 min at 37 °C and 5% CO2. Cells were washed twice in cold 1× PBS, stained with surface antibodies and analyzed by FACS. For confocal microscopy, cells were stained at 37 °C for 30 min with 300 ng ml–1 of Hoechst H33342 (Life Technologies) to stain nuclei, 100 nM MitoTracker Green FM to stain mitochondria and 25 nM TMRM to asses mitochondrial membrane potential (non-quenching mode, TMRM maintained in the cell medium). Cells were plated in a 384-well plate (40,000 cells per well), and image acquisitions of multiple fields per well were performed on an automated confocal microscope (OPERA QEHS, Perkin Elmer) using ×60 objectives, excitation lasers at 405, 488 and 561 nm and emission filters at 450, 540 and 600 nm, respectively. Confocal images were transferred to the Columbus Image Data Storage and Analysis System (Perkin Elmer) for high content analyses as previously reported57 (link) and used the standard deviation/mean approach58 (link).
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