Isolated neutrophils were suspended in a culture medium containing Rosewell Park Memorial Institute 1640, antibiotics (100 U penicillin/ml and 50 ng streptomycin/ml), and serum (4%). They were stimulated to form NETs on 96-well culture plates. For this purpose, the cells were introduced in an amount of 5 × 104 per well. Then, they were incubated for 60 min at 37 °C under 5% CO2 (Nuarie™ US Autoflow) in the absence or presence of LPS (10 μM), BPA (16 nM or 1.6 μM), or E2 (100 pM). Hoechst 3342 dye (Invitrogen) (at a concentration of 1 μg/ml prepared in PBS), which allows the detection of DNA, and antihuman MPO antibody (Life Technologies), which allows detecting the main NET component, were added to the samples. The analysis of NET formation in the wells was carried out under a fluorescence microscope (IN Cell Analyzer 2200, GE Healthcare Life Sciences) using the IN Cell Analyzer Workstation program. The stained cells were counted in nine 20× microscopic fields per section. The results were expressed as the percentage of cells showing the NET formation related to all the neutrophils of an image. For statistical analysis, the mean value of nine images was used for calculating the average values for each sample.
Hoechst 3342 dye
Manufactured by Thermo Fisher Scientific
Sourced in United States, Canada
Hoechst 3342 dye is a fluorescent DNA-binding compound. It is used in various laboratory applications to stain and visualize DNA. The dye binds to the minor groove of DNA and emits fluorescence upon excitation, allowing for the detection and analysis of DNA molecules.
Automatically generated - may contain errors
Lab products found in correlation
In cell analyzer 2200, by GE Healthcare (3 mentions)
Antihuman mpo antibody, by Thermo Fisher Scientific (3 mentions)
Prolong gold antifade mounting medium, by Thermo Fisher Scientific (2 mentions)
A1si spectral detector confocal system, by Nikon (1 mentions)
Alexa fluor 647 phalloidin, by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
Dylight549 labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
Hif 1α, by Cell Signaling Technology (1 mentions)
β catenin, by Cell Signaling Technology (1 mentions)
Hct116, by American Type Culture Collection (1 mentions)
Sw480, by American Type Culture Collection (1 mentions)
7 protocols using hoechst 3342 dye
1
Neutrophil Extracellular Trap Formation Assay
2
Visualizing Osteoclast F-actin Ring Formation
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In order to observe the F-actin ring (the ruffled membrane of an osteoclast) formation, we cultivated the BMM-derived osteoclasts on bovine bone slices, and then treated them with different Pra-C concentrations (0, 5, 10, or 20 μM) for 48 h. Following that, we fixed the cells with 4% paraformaldehyde for 20 min. The cells were then permeabilized with 0.1% (v/v) Triton X-100 (Sigma-Aldrich, St. Louis, MO, United States) for 5 min, and incubated with Alexa Fluor 647 phalloidin (Invitrogen, San Diego, CA, United States) for 1 h. The cells were washed with PBS three times. The cell nuclei were then stained with Hoechst 3342 dye (1:5000; Invitrogen), washed with PBS, and mounted on microscope slides with ProLong Gold anti-fade mounting medium (Invitrogen). Fluorescent staining of the F-actin ring was observed with a NIKON A1Si spectral detector confocal system with 10× (dry) lenses equipped.
3
Osteoclast F-actin Ring Imaging
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For F-actin ring immunofluorescent staining, WT and PIP5k1β −/− osteoclasts were fixed with 4% PFA for 15 min at room temperature and permeabilized for 5 min with 0.1% (v/v) Triton X-100 on ice. Cells were incubated with fluorescent phalloidins (1:40; Invitrogen Life Technologies) diluted in 1% (w/v) bovine serum albumin-PBS for 20 min at room temperature and then washed extensively with PBS. Cells were then incubated with Hoechst 3342 dye (1:5000; Invitrogen Life Technologies) for visualizing nuclei, washed with PBS, and mounted with ProLong Gold anti-fade mounting medium (Invitrogen Life Technologies). Fluorescence was detected with NIKON A1Si spectral detector confocal system equipped with 20 (dry) lenses. Fluorescence images were obtained with NISeC Elements software (National Institutes of Health).
4
Neutrophil Extracellular Trap (NET) Formation
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Isolated neutrophils were suspended in a culture medium containing Rosewell Park Memorial Institute 1640, antibiotics (100 U penicillin/ml and 50 ng streptomycin/ml), and serum (4%). They were stimulated to form NETs on 96-well culture plates. For this purpose, the cells were introduced in an amount of 5 × 10 4 per well. Then, they were incubated for 60 minutes at 37°C under 5% CO 2 (Nuarie™ US Auto ow) in the absence or presence of LPS (10 µM), BPA (16 nM or 1.6 µM), or E2 (100 pM). Hoechst 3342 dye (Invitrogen) (at a concentration of 1 µg/ml prepared in PBS), which allows the detection of DNA, and antihuman MPO antibody (Life Technologies), which allows detecting the main NET component, were added to the samples.
The analysis of NET formation in the wells was carried out under a uorescence microscope (IN Cell Analyzer 2200, GE Healthcare Life Sciences) using the IN Cell Analyzer Workstation program. The stained cells were counted in nine 20× microscopic elds per section. The results were expressed as the percentage of cells showing the NET formation related to all the neutrophils of an image. For statistical analysis, the mean value of nine images was used for calculating the average values for each sample.
The analysis of NET formation in the wells was carried out under a uorescence microscope (IN Cell Analyzer 2200, GE Healthcare Life Sciences) using the IN Cell Analyzer Workstation program. The stained cells were counted in nine 20× microscopic elds per section. The results were expressed as the percentage of cells showing the NET formation related to all the neutrophils of an image. For statistical analysis, the mean value of nine images was used for calculating the average values for each sample.
5
Neutrophil Extracellular Trap Formation Assay
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Isolated neutrophils were suspended in a culture medium containing Rosewell Park Memorial Institute 1640, antibiotics (100 U penicillin/ml and 50 ng streptomycin/ml), and serum (4%). They were stimulated to form NETs on 96-well culture plates. For this purpose, the cells were introduced in an amount of 5 × 10 4 per well. Then, they were incubated for 60 minutes at 37°C under 5% CO 2 (Nuarie™ US Auto ow) in the absence or presence of LPS (10 µM), BPA (16 nM or 1.6 µM), or E2 (100 pM). Hoechst 3342 dye (Invitrogen) (at a concentration of 1 µg/ml prepared in PBS), which allows the detection of DNA, and antihuman MPO antibody (Life Technologies), which allows detecting the main NET component, were added to the samples. The analysis of NET formation in the wells was carried out under a uorescence microscope (IN Cell Analyzer 2200, GE Healthcare Life Sciences) using the IN Cell Analyzer Workstation program. The stained cells were counted in nine 20× microscopic elds per section. The results were expressed as the percentage of cells showing the NET formation related to all the neutrophils of an image. For statistical analysis, the mean value of nine images was used for calculating the average values for each sample.
6
Colorectal Cancer Cell Line Cultures
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CRC cell lines HCT116 and SW480 were obtained from the American Type Culture Collection (ATCC). Cell lines were cultured in ATCC-recommended media supplemented with 10% fetal bovine serum (FBS) at 37°C and 5% CO2. Antibodies directed against pAKT (S473), pS6 (S235/236), p4EBP1 (Thr 37/46), pERK (Y202/204), Bcl-2, β-catenin, HIF-1α, cyclin D1, and actin were obtained from Cell Signaling Technology (Danvers, MA, USA). Dylight549-labeled secondary antibody and Hoechst3342 dye were obtained from Thermo Fischer Scientific (Waltham, MA, USA). Compounds sorafenib, BEZ-235, and GDC-0941 were obtained from AXON chemicals (Reston, VA, USA).
7
Hoechst Assay for Apoptosis Detection
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Hoechst assay was used to detect apoptotic cells in CF, MF and cardiomyocytes. Twenty-four hours after Rsv treatment, CF, MF and cardiomyocytes were incubated with 1.5 µL/well Hoechst 3342 dye (10 mg/mL; ThermoFisher, Ottawa, ON, Canada) for 20 min in CO2 incubator. Cells were then imaged using EVOSfl fluorescent microscope at 10X magnification. The images were analyzed using ImageJ software.
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