For drug treatment, lung cancer cells (NCI-H460, A549, and SK-MES-1) and stromal cells (WI38 and HUVEC) were seeded at a density of 6 × 103 cells/well in 96-well round-bottomed ultra-low attachment microplates. After 1 day, 10 or 20 μM of Gefitinib and Cisplatin (all from Sigma-Aldrich, St. Louis, MO, USA) were added to the spheroids for 2 days, and then spheroid cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen Life Technologies, Grand Island, NY, USA). EthD-1 is a high-affinity nucleic acid stain that fluoresces weakly until bound to DNA, whereupon it emits red fluorescence (excitation/emission maxima ~ 528/617 nm). Spheroids were incubated in 4 μM EthD-1 in complete medium for 30 min in a 37 °C incubator, and images were obtained and the intensity of EthD-1 fluorescence measured using the Operetta® High Content Screening System (Perkin Elmer).
Ethd 1
Manufactured by PerkinElmer
Sourced in United States
EthD-1 is a fluorescent dye used for nucleic acid detection and quantification. It intercalates with DNA and RNA, emitting fluorescence upon binding. The dye is commonly used in various applications such as gel electrophoresis and flow cytometry to visualize and quantify nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Operetta high content screening system, by PerkinElmer (3 mentions)
Cisplatin, by Merck Group (3 mentions)
Gefitinib, by Merck Group (2 mentions)
Harmony software, by PerkinElmer (2 mentions)
Ethd 1, by Thermo Fisher Scientific (2 mentions)
96 well round bottomed ultra low attachment microplates, by Corning (2 mentions)
Ethidium homodimer 1 ethd 1, by Thermo Fisher Scientific (1 mentions)
3 protocols using ethd 1
1
Assessing Drug-Induced Spheroid Cell Death
2
Assessing Lung Cancer Spheroid Death
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Non-specific siRNA (siCont) or HYOU1 siRNA (siHYOU1)transfected for 24 h or 5, 10, 20, or 40 μM of gefitinib and cisplatin (all from Sigma-Aldrich, USA) treated for 48 h, lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) with or without HUVECs were seeded at a density of 6 × 103 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning). After 2 or 3 days, the spheroids cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen). EthD-1 is a high-affinity nucleic acid stain that fluoresces weakly until bound to DNA, whereupon it emits red fluorescence (excitation/emission maxima ~528/617 nm). Spheroids were incubated in 4 μM EthD-1 in complete medium for 30 min in a 37°C incubator, and images were obtained and the intensity of EthD-1 fluorescence measured using the Operetta® High Content Screening System (Perkin Elmer, USA). Fluorescent intensity analysis was performed using the Harmony software (Perkin Elmer).
3
Spheroid Cell Death Assay for Lung Cancer
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Non-speci c SiRNA (SiCont) or HYOU1 SiRNA (SiHYOU1) transfected for 24 h or 5, 10, 20, or 40 µM of ge tinib and cisplatin (all from Sigma-Aldrich, St. Louis, MO, USA) treated for 48 h, lung cancer cells (NCI-H460 cells, A549 cells, or H1299 cells) with or without HUVECs were seeded at a density of 6 × 10 3 cells/well in 96-well round-bottomed ultra-low attachment microplates (Corning, NY, USA). After 2 or 3 days, the spheroids cell death was detected using the cell-impermeant viability indicator ethidium homodimer-1 (EthD-1; Invitrogen, Eugene, OR, USA). EthD-1 is a high-a nity nucleic acid stain that uoresces weakly until bound to DNA, whereupon it emits red uorescence (excitation/ emission maxima ~ 528/617 nm). Spheroids were incubated in 4 µM EthD-1 in complete medium for 30 min in a 37 °C incubator, and images were obtained and the intensity of EthD-1 uorescence measured using the Operetta® High Content Screening System (Perkin Elmer, Waltham, MA, USA). Fluorescent intensity analysis was performed using the Harmony software (Perkin Elmer, Waltham, MA, USA).
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