Cells were seeded in Greiner 96‐well microtiter plates (Greiner Bio‐One), fixed in 4% paraformaldehyde for 10 min, and then incubated in 0.5% Triton X‐100 in PBS for 10 min at room temperature as previously reported 7 . After the cells were blocked in 5% fetal bovine serum/TPBS for 1 h at room temperature, they were incubated overnight at 4 °C with primary antibodies, including mouse anti‐TG2 (1 : 200, ab2386; Abcam, Cambridge, MA, USA), rabbit anti‐nuclear factor erythroid‐2‐related factor 2 (Nrf2) (1 : 400, ab62352; Abcam), mouse anti‐Ki67 (1 : 200, 350502; BioLegend, San Diego, CA, USA), or control rabbit/mouse IgG. The cells were then washed and stained with fluorochrome (FITC/TRITC)‐conjugated secondary antibodies (1 : 500; Invitrogen, Eugene, OR, USA) for 20 min at room temperature. Cell nuclei were visualized using DAPI (1 : 2000; Wako Industries). Cellular fluorescence signals were detected using an ImageXpressMICRO High‐Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using the ‘Multi‐Wavelength Cell Scoring Application Module’ in MetaXpress Image Analysis software (Molecular Devices).
Multi wavelength cell scoring application module
Manufactured by Molecular Devices
The Multi-Wavelength Cell Scoring Application Module is a laboratory equipment designed to analyze and quantify cells. It utilizes multiple wavelengths to detect and measure various cellular characteristics. The core function of this module is to provide accurate and reliable data on cell populations without interpretation or extrapolation.
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Lab products found in correlation
96 well microtiter plate, by Greiner (2 mentions)
Imagexpress micro high content screening system, by Molecular Devices (2 mentions)
Metaxpress image analysis software, by Molecular Devices (2 mentions)
Metaxpress software, by Molecular Devices (2 mentions)
Ab2386, by Abcam (1 mentions)
Ab62352, by Abcam (1 mentions)
Dmi6000, by Leica (1 mentions)
Volocity software, by PerkinElmer (1 mentions)
Hoechst 33342 dye, by Thermo Fisher Scientific (1 mentions)
Imagexpress micro xl high content imager, by Molecular Devices (1 mentions)
4 protocols using multi wavelength cell scoring application module
1
High-Content Screening of Cellular Markers
2
Measuring Intracellular Transglutaminase 2 Activity
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Intracellular TG2 transamidase activity was measured as previously described [34 (link)]. Briefly, the cells were seeded in Greiner 96-well microtiter plates (Greiner Bio-One, Kremsmuenter, Austria) and incubated with 0.2 mM 5BAPA and 0.1 mM aminoguanidine overnight at 37 °C. The cells were treated with increasing concentrations of ACR in serum-free media for 4 h and then fixed with a 10% formaldehyde solution, permeabilized, blocked, and stained with streptavidin-tetramethylrhodamine isothiocyanate (TRITC, Jackson ImmunoResearch Laboratories). Intracellular TG2 transamidase activity was then detected as a fluorescence signal from TRITC and analyzed using an ImageXpressMICRO High Content Screening System (Molecular Devices, Sunnyvale, CA, USA). Morphological analysis was performed using a Multi-Wavelength Cell Scoring Application Module in MetaXpress Image Analysis software (Molecular Devices).
3
Live-Cell Imaging for Neurite Quantification
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Live-cell imaging was performed on the automated image acquisition fluorescence microscope system ImageXpress (Molecular Devices) using a 20x magnification with DAPI, FITC, and Texas Red filter cubes at multiple positions. GFP reporter fluorescence was analyzed using the MetaXpress software and the multi-wavelength cell scoring application module (Molecular Devices). Long-term time-lapse imaging was performed on the fluorescence microscope system DMI6000 (Leica) at 37 °C and 5% CO2 using a 20x magnification with DIC, mCherry, and GFP filter sets at 4 positions per condition. Images were acquired at 10 min intervals. Images were analyzed using Volocity software (Perkin Elmer). For neurite quantification, a neurite was defined as a process extending from the soma by at least one cell diameter (10 μm). Cell viability was determined based on the relative number of pyknotic or fragmented nuclei following incubation with Hoechst 33342 dye (Life Technologies).
4
Automated High-Content Cellular Imaging
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Imaging of the immunofluorescence-stained cultures was performed with Molecular Devices’ ImageXpress Micro XL high-content imager. Briefly, the post-laser z-offset was determined for correct autofocusing, and the exposure time for each illumination filter was calculated. Several wells across the 384-well plate were tested for consistency prior to acquisition of the entire plate.
Analysis of the fluorescent images was done with Molecular Devices’ MetaXpress software and their Multi-Wavelength Cell Scoring Application Module. Briefly, the minimum and maximum widths as well as the signal intensity above local background were determined for proper segmentation of the nuclear Hoechst 33342 stain and the cytoplasmic CK8/18 stain (entire cell). Several wells of the 384-well plate were previewed by eye for accurate segmentation prior to analysis of the entire plate. Data collected from the analysis included the number of total cells (Hoechst 33342-positive nuclei count), the number of epithelial cells (Hoechst 33342-positive and CK8/18-positive cell count), and the number of non-epithelial cells (Hoechst 33342-positive and CK8/18-negative cell count).
Analysis of the fluorescent images was done with Molecular Devices’ MetaXpress software and their Multi-Wavelength Cell Scoring Application Module. Briefly, the minimum and maximum widths as well as the signal intensity above local background were determined for proper segmentation of the nuclear Hoechst 33342 stain and the cytoplasmic CK8/18 stain (entire cell). Several wells of the 384-well plate were previewed by eye for accurate segmentation prior to analysis of the entire plate. Data collected from the analysis included the number of total cells (Hoechst 33342-positive nuclei count), the number of epithelial cells (Hoechst 33342-positive and CK8/18-positive cell count), and the number of non-epithelial cells (Hoechst 33342-positive and CK8/18-negative cell count).
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