A549 or MeT‐5A cells were cultured to 70%‐80% confluence in six‐well plates, treated with asbestos and/or zeolites for 24‐36 hours, as in the viability assays, then treated with CellROX Orange (Life Technologies) using the supplier's protocol, followed by measurement of ROS fluorescence in a Tali Image‐Based Cytometer using the same number of fields and pooled cells as in the viability assays. CellROX reagents are fluorogenic probes for measuring generalized oxidative stress in cells, and were used here to measure levels of ROS in response to different treatments. While reagents selective for a variety of reactive species targeted to the cytosol or mitochondria are available, these were considered beyond the scope of the current study.
Image based cytometer
Manufactured by Thermo Fisher Scientific
The Image-based cytometer is a laboratory instrument used for the analysis and quantification of cells, particles, and other biological samples. The core function of this device is to capture and analyze high-resolution images of individual cells or particles, providing detailed information about their size, shape, and other morphological characteristics.
Automatically generated - may contain errors
3 protocols using image based cytometer
1
Oxidative Stress Response to Asbestos and Zeolites
2
Omomyc Induces Apoptosis in A549 Cells
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Example 4
Omomyc Reduces the Number of Viable A549 Cells and Induces Apoptosis
A549 cells were incubated in 24-well plates over a period of 72 hours with 10 μM Max* or Omomyc. Max* was obtained as described in Montagne M. et al. (Montagne M. et al. 2012. PLoS One, 7(2):e32172). Cells were harvested and stained with Annexin V and PI according to the manufacturer protocol (Tali® Apoptosis Kit A10788, Life technologies) and the fluorescence quantified using Tali® image-based cytometer.
In this study the authors of the present invention show that Omomyc is more efficient at reducing the number of viable A549 cells than the bHLHZ domain of Max (
3
Apoptosis Quantification via Microscopy
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Apoptosis analyzes were performed 72 hours post-exposure of cells to various treatments, including LP, LP-HA, LP-Quer, LP-Quer-HA, and 10 µM Quer. Utilizing specific kits (Life Technologies), the percentage of viable, dead and apoptotic cells was determined employing an image-based cytometer (Life Technologies) [27 (link)]. Each group underwent four separate measurements, and the experiments were repeated three times.
Additionally, apoptosis in cells treated as described above was identified via Hoechst 33342 staining. Images were captured at 340 × 510 nm using a fluorescence microscope (×40) (Axio Vert A1; ZEISS).
Additionally, apoptosis in cells treated as described above was identified via Hoechst 33342 staining. Images were captured at 340 × 510 nm using a fluorescence microscope (×40) (Axio Vert A1; ZEISS).
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