Formalin-fixed, paraffin embedded left lung lobe sections (6 µm) were used for immunohistochemical staining for α-smooth muscle actin (α-SMA). Sections were deparaffinized with xylene (2 × 5 min each) and rehydrated with graded ethanol (100%, 90%, 70%, and tap water). Antigen retrieval was performed with 10 mM sodium citrate at 100°C for 1 h. The slides were blocked for 1 h in TBS containing 4% goat serum and 0.04% Triton X-100. After washing, slides were incubated overnight in monoclonal mouse anti-α-SMA at 1:100 dilution (Millipore Sigma, St. Louis, MO, Cat. No. A2547). After incubation, slides were washed and incubated with anti-mouse-FITC secondary antibody (Millipore Sigma, Cat. No. F0257) at 1:1,000 dilution. Negative control slides followed the same protocol but without addition of a primary antibody. Slides were counterstained with DAPI, and images taken at ×100 using Lionheart (Biotek, Winooski, VT). Smooth muscle actin area was analyzed using ImageJ (NIH). Data were normalized to length of airway basement membrane to report as airway smooth muscle (ASM) mass per µm basement membrane.
Lionheart
Manufactured by Agilent Technologies
Sourced in United States
The Lionheart is a high-performance automated cell imaging system designed for cell-based assays and live-cell analysis. It captures high-quality images and time-lapse videos of cells in a variety of plate formats.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (2 mentions)
Alexa fluro 488, by Thermo Fisher Scientific (1 mentions)
Ix83 microscope, by Olympus (1 mentions)
A2547, by Merck Group (1 mentions)
Fluorescent reader, by Agilent Technologies (1 mentions)
Mitosox red, by Agilent Technologies (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Premiere pro, by Adobe (1 mentions)
Matrigel matrix, by Corning (1 mentions)
Alkaline phosphatase kit, by Merck Group (1 mentions)
Biotinylated ha binding protein, by Merck Group (1 mentions)
22 protocols using lionheart
1
Quantifying Airway Smooth Muscle Mass
2
Quantifying Nrf2 Phosphorylation in Cells
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The cells were cultured under a sterile coverslip until 60–70% confluency in 6-well culture plates and then treated with SeC for 24 h. They were fixed and permeabilized using methanol. Anti-Nrf2 (phospho S40, ARG40667, 225×) antibody was obtained from Arigo Biolaboratories. Anti-rabbit IgG conjugated with Alexa Fluro 488 (Invitrogen, Molecular Probe) was used as the secondary antibody, and propidium iodide (PI) was used as the nuclear counterstain. Nrf2 phosphorylation at Ser40 was determined using a fluorescence scanner (Lionheart; BioTeK). The cell count in each field was determined using Image J.
3
Microscopic Cell-Cell Junction Imaging
4
Quantifying Airway Smooth Muscle Mass
5
Measuring Cellular Oxidative Stress Levels
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The cells were added to 96-well black plates. After the indicated treatment, 25 μM H2DCF-DA (Sigma-Aldrich) was added to the wells. After 45 min, fluorescence was detected at Ex 485 nm/Em 525 nm on a fluorescent reader from BioTeK. The mitochondrial and cellular superoxide levels were measured using MitoSOX Red (Invitrogen, Molecular Probe, OR, USA) and DHE (Invitrogen).
The cells were cultured under a sterile coverslip until 60–70% confluency in a six-well culture plate. After SeC treatment, the cells were stained with 2 μM MitoSOX or DHE at 37 °C for 30 min. The cells were then washed three times with phosphate-buffered saline (PBS) to remove excess fluorescent dye; the fluorescence intensity was then determined using a fluorescence scanner (Lionheart; BioTeK) or Guava Muse Cell Analyzer at 580 nm for MitoSOX Red and 600 nm for DHE. The average fluorescent intensity was calculated as MitoSOX Red or DHE intensity divided by cell count in the field. The cell count in each field was determined using Image J.
The cells were cultured under a sterile coverslip until 60–70% confluency in a six-well culture plate. After SeC treatment, the cells were stained with 2 μM MitoSOX or DHE at 37 °C for 30 min. The cells were then washed three times with phosphate-buffered saline (PBS) to remove excess fluorescent dye; the fluorescence intensity was then determined using a fluorescence scanner (Lionheart; BioTeK) or Guava Muse Cell Analyzer at 580 nm for MitoSOX Red and 600 nm for DHE. The average fluorescent intensity was calculated as MitoSOX Red or DHE intensity divided by cell count in the field. The cell count in each field was determined using Image J.
6
Huh-7 and Hep3B Cell Proliferation Assay
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Huh-7 and Hep3B cells were seeded into 96-well plates (2.5×104 cells/well) and incubated at 37°C until the cells reached 30% confluence. Next, the cells were incubated with EdU (50 µM) for 2 h, 0.5% Triton X-100 and ApolloR reaction cocktail (100 µl) for 30 min and 100 µl Hoechst 33342 for 30 min, sequentially. Cell proliferation was analyzed by assessing the percentage of EdU-positive cells in all cells in each sample using a fluorescence microscope (Lionheart; BioTek Instruments, Inc.; magnification, ×100) and ImageJ software (version 1.8.0; National Institutes of Health) was used for cell counting.
7
Mitosis Quantification in Monolayer Cells
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To evaluate the occurrence of mitosis in monolayer cells under the conditions mentioned in Section 2.4 , the cells were fixed with 3.7% formaldehyde in PBSA after 24 h of the exposition of the GOX materials, the plasma membrane was permeabilized with 0.5% Triton X-100 for 20 min. After that, they were incubated overnight with a primary monoclonal anti-α tubulin antibody inside a moist chamber. On the following day, after three washes with PBSA, the cells were incubated for 2 h with the fluorochrome-conjugated secondary anti-mouse antibody. Subsequently, the nuclei were stained with Hoechst 33258 (Sigma, São Paulo, Brazil) for 30 min. Finally, the cytological preparations were mounted on the microscopic slide with Vectashield (Vector). Under the Lionheart (Biotek, Winooski, VT, USA) microscope, the nucleus area was determined and mitosis was quantified in at least 6 fields by preparations.
8
Cell Proliferation Assessment by EdU Assay
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Cell proliferation was assessed by use of an EdU assay kit (Ruibo Biotechnology Co., Ltd., Guangzhou, China). Tumor cells (1×105 cells/well) were incubated with 100 µl of 50 µM EdU per well for 2 h at 37°C in 24-well plates, and then were fixed with 100 µl of fixing buffer (4% polyformaldehyde in PBS) for 30 min at room temperature. Subsequently, the cells were incubated for 5 min with 50 µl of 2 mg/ml glycine, and washed with 100 µl PBS. Next, the cells were treated with 0.5% Triton X and reacted with 1X Apollo solution for 30 min. The cells were then incubated with 100 µl Hoechst 33342 solution for 30 min in the dark, and washed with 100 µl PBS. Finally, the cells were analyzed by fluorescence microscopy (Lionheart; BioTek Instruments, Inc., Winooski, VT, USA).
9
Long-term 3D Organoid Imaging
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For 3D imaging, the HGSC organoids were embedded in 45% phenol red–free Matrigel matrix (356231, Corning) mixed with 55% advanced imaging medium on a glass-bottom culture plate (0.16–0.19 mm thick) (801004, NEST). For a six-well plate, 4 ml of advanced imaging medium was added to each well to support organoid growth during imaging.
A Lionheart (Biotek) multifunction imaging instrument was used to perform long-term time-lapse live-cell imaging. The key optical components include DAPI cube assembly (LED: 1225007/Filter Cube: 1225100); GFP cube assembly (LED: 1225001/Filter Cube: 1225101); GFP cube assembly (LED: /Filter Cube:); Texas Red cube assembly (LED: 1225002/Filter Cube: 1225102).
Gen5+ software was applied for image acquisition and instrument control. The raw images were further processed in Fiji software (version 1.52p) (RRID: SCR_002285), Adobe Photoshop (RRID: SCR_014199), Adobe Illustrator (RRID: SCR_010279), and Adobe Premiere Pro (RRID: SCR_021315), for generating time-lapse videos and figures.
A Lionheart (Biotek) multifunction imaging instrument was used to perform long-term time-lapse live-cell imaging. The key optical components include DAPI cube assembly (LED: 1225007/Filter Cube: 1225100); GFP cube assembly (LED: 1225001/Filter Cube: 1225101); GFP cube assembly (LED: /Filter Cube:); Texas Red cube assembly (LED: 1225002/Filter Cube: 1225102).
Gen5+ software was applied for image acquisition and instrument control. The raw images were further processed in Fiji software (version 1.52p) (RRID: SCR_002285), Adobe Photoshop (RRID: SCR_014199), Adobe Illustrator (RRID: SCR_010279), and Adobe Premiere Pro (RRID: SCR_021315), for generating time-lapse videos and figures.
10
Wound Healing Assay in Cell Cultures
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Cells were seeded in 6-well plates. After the cells reached confluence, a wound was made with a 200-µL plastic tip in each well. The wells were then washed twice with PBS to remove cell debris and then incubated with a culture medium. The wound areas were automatically monitored for 24 h by taking a photograph every 30 min in an augmented microscope (Lionheart, BioTek, Winooski, VT, USA). The wound areas were automatically analyzed by computer, and the wound healing curve was performed based on the area change over time. The wound area of each condition was presented as 100 at the beginning. The values are the mean of four wounds in each well.
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