NHBE cells were seeded into black, clear-bottom 96-well tissue culture plates at a density of 37,500 cells per cm2 (12,000 cells per well) in 100 μL of culture medium. The cells were incubated for 24 h in the culture medium and then exposed (in three replicate wells) to increasing concentrations of the e-liquid solutions containing the different flavoring substances. A corresponding base solution was included as a reference. The cells were exposed for 30 min (NF-κB endpoint only), 4 h, and 24 h before performing the HCS assays. In parallel, appropriate positive controls at three different concentrations were used for each endpoint (Table S2). Dimethyl sulfoxide (CASRN 67-68-5; purity >99.9%; ref. D8418, Sigma-Aldrich) was used as the vehicle for all positive control treatments at a final concentration of 0.5%. Cell count was recorded in all assays using Hoechst 33342 staining (Life Technologies, Carlsbad, CA, USA), except for the oxidative stress assay, where DRAQ5™ dye (Biostatus, Shepshed, UK) was used. Following staining of the NHBE cells, fluorescence was analyzed by image acquisition with a Thermo Fisher Cellomics® ArrayScan VTI High Content Screening Reader (Thermo Fisher Scientific, Waltham, MA, USA) and vHCS™:View software (Thermo Fisher Scientific). Twenty fields were imaged per well using a 10× wide field objective, as previously described [15 ].
Draq 5 dye
Manufactured by Biostatus
Sourced in United Kingdom, United States
DRAQ-5 is a fluorescent dye used for staining and detection of DNA in biological samples. It binds to DNA and emits fluorescence when excited by an appropriate light source, allowing visualization and analysis of DNA content in cells or tissues.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade reagent, by Thermo Fisher Scientific (5 mentions)
Lsm 5 exciter confocal laser scanning microscope, by Zeiss (3 mentions)
Plan neofluar 40 1.3 na dic, by Zeiss (2 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Hoechst 33342 staining, by Thermo Fisher Scientific (1 mentions)
Lsm 5 exciter, by Zeiss (1 mentions)
α tubulin antibody, by Cell Signaling Technology (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit antibody, by Thermo Fisher Scientific (1 mentions)
Zen lite software, by Zeiss (1 mentions)
Rabbit anti p27 antibody, by Abcam (1 mentions)
Glass chamber slides, by Sarstedt (1 mentions)
Anti orai1 primary antibody, by Abcam (1 mentions)
Cf 488a labeled anti rabbit secondary antibody, by Merck Group (1 mentions)
6 protocols using draq 5 dye
1
High-Content Screening of E-Liquid Toxicity
2
Chorein Localization in ZF and CaCo2 Cells
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For chorein staining, ZF and CaCo2 cells were cultured on glass cover slips for 24 h. After washing twice with PBS, cells were fixed with 4% PFA for 15 min on RT and then permeabilized with 0,03% Triton-X100 for 10 min. After blocking with 3% BSA in PBST cells were incubated at 4°C overnight with anti-VPS13A-antibody (1:300, Sigma-Aldrich, Germany). The cells were rinsed three times with PBST and incubated with secondary goat anti-rabbit CFTM 488 antibody (1:300, Sigma-Aldrich, Germany) for 2 h at room temperature. Additional cells were incubated 30 min in the dark with rhodamine-phalloidin (1:200, Life Technologies, USA) for F-actin staining and with DRAQ-5 dye (1:3000, Biostatus, Leicestershire, UK) for nuclei staining. After three washing steps all slides and coverslips were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 63/1.3 NA DIC.
3
Immunostaining of Microtubules
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Concisely, cells stayed in PBS at 5 ×107 cells/ml. Of which, 10 µl of sample was taken on glass slide and dried for 30 min before fixation with methanol. After repeated PBS washing for 10 min, the sample was utilized. To look for microtubular staining, cells were fixated with 4% PFA at room temperature for 15 min. Another dual wash was performed with PBS. The slides were then incubated with blocking buffer (1xPBS/5% normal goat serum/0.3% Triton™ X-100) for 60 min at room temperature. Next, the samples were subjected to α-tubulin antibody overnight at temperature 4 oC (1:50; Cell Signaling). The samples were then washed three times with PBS and incubated for 90 min with Goat anti-Rabbit IgG (FITC) antibody (1:500; Invitrogen). Next, nuclei were dyed for 10 min using DRAQ-5 dye (Biostatus). Mounting was executed through ProLong® Gold Antifade reagent (Invitrogen). Lastly, Zeiss LSM 5 EXCITER was used for confocal microscopy and images were examined using the same instrument-embedded software.
4
Visualizing Na/K ATPase Subunit in DCs
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For visualization of the α1 subunit of Na/K ATPase in DCs from jak3 -/-and jak3 +/+ mice, confocal microscopy was performed. Cultured DCs were seeded at 10 5 on Poly-L-Lysine (Sigma-Aldrich, Germany) coated chamber slides.
After 4 h, the attached cells were fixed with 4% PFA for 15 min at RT and permeabilized with 0.03% Triton-X100 for 10 min at RT. After blocking with 3% BSA in PBST cells were incubated at 4°C overnight with anti-ATP1A1antibody (1:100, Cell Signaling). The cells were rinsed three times with PBST and incubated with secondary goat anti-rabbit CF™ 488 antibody (1:300, Sigma-Aldrich, Germany) for 2h at room temperature. In addition cells were incubated for 30 min in the dark with DRAQ-5 dye (1:4000, Biostatus, Leicestershire, UK) for nuclei staining. After three washing steps, the slides were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC. Negative controls were carried out by incubation in absence of primary antibody.
After 4 h, the attached cells were fixed with 4% PFA for 15 min at RT and permeabilized with 0.03% Triton-X100 for 10 min at RT. After blocking with 3% BSA in PBST cells were incubated at 4°C overnight with anti-ATP1A1antibody (1:100, Cell Signaling). The cells were rinsed three times with PBST and incubated with secondary goat anti-rabbit CF™ 488 antibody (1:300, Sigma-Aldrich, Germany) for 2h at room temperature. In addition cells were incubated for 30 min in the dark with DRAQ-5 dye (1:4000, Biostatus, Leicestershire, UK) for nuclei staining. After three washing steps, the slides were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC. Negative controls were carried out by incubation in absence of primary antibody.
5
Immunofluorescent Labeling of Cardiomyocytes
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HL-1 cardiomyocytes were fixed with 4% paraformaldehyde/PBS for 15 min at RT and permeabilized with PBS/0.1% Triton-X for 10 min at RT. After blocking with 5% normal goat serum in PBS/0.1% Triton-X for 1 hour at RT, the slides were incubated overnight at 4°C with rabbit anti-p27 antibody (diluted 1:50, Abcam) or with rabbit anti-SGK1 antibody (diluted 1:50, Pineda) and then with goat anti-rabbit Alexa Fluor488-conjugated antibody (diluted 1:1,000, Invitrogen) for 1 hour at RT. Nuclei were stained using DRAQ-5 dye (diluted 1:1000, Biostatus) and actin using Rhodamine Phalloidin (diluted 1:100, Invitrogen). The slides were mounted with Prolong Gold antifade reagent (Invitrogen). Images were collected with a confocal laser-scanning microscope (LSM 510, Carl Zeiss MicroImaging GmbH) using a water immersion A-Plan ×63/1.2W. Negative controls were carried out simultaneously by omitting incubation with primary antibodies. Cell size was quantified by measuring the area of attached cells in Rhodamine Phalloidin stained HL-1 cardiomyocytes with Zen Lite software (Zeiss). 30 random cells were analyzed in 5-6 pictures of each biological replicate [41] (link).
6
Orai1 Localization in Chorein-Silenced Cells
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For confocal laser scanning microscopy negative and chorein silenced ZF cells were seeded on glass chamber slides (Sarstedt, Germany). After washing twice with PBS, cells were fixed with 4% PFA for 15 min and blocked with 3% BSA in PBS for 1 hour at room temperature. Then, the cells were exposed to anti-Orai1 primary antibody (1:200, Abcam) at 4 °C overnight. After three washing steps with PBS the cells were incubated with CF™ 488A-labeled anti-rabbit secondary antibody (1:250, Sigma, USA) and with DRAQ-5 dye (1:3000, Biostatus, Leicestershire, UK) for 1 h at room temperature. Following three washes with PBS all slides were mounted with ProLong Gold antifade reagent (Life Technologies, USA). Images were subsequently taken on a Zeiss LSM 5 EXCITER confocal laser scanning microscope (Carl Zeiss, Germany) with a water immersion Plan-Neofluar 40/1.3 NA DIC [14, 34] .
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