The mitochondrial health assay (H10295, Thermo Fisher Scientific) utilizes a combination of three dyes to assess mitochondrial functionality: a fluorescent dye that accumulates in mitochondria in a membrane potential-dependent manner (used 1:700), image-IT green to identify dead cells (1:2000), and Hoescht blue nuclear stain (1:2000). Live cells were treated with the dye for 30 minutes while being maintained in standard culture conditions. Images and quantification were obtained using the Cellinsight Cx7 (Thermofisher, CA, USA). Cells grown in standard culture conditions were also treated with 5μM CellROX (C10422, Thermo Fisher Scientific) for one hour, then washed, fixed, and counterstained with Hoescht for imaging and quantification on the Cellinsight Cx7.
Cellinsight cx7
Manufactured by Thermo Fisher Scientific
Sourced in United States
The CellInsight CX7 is a high-content analysis imaging system designed for cellular and subcellular imaging. It provides automated image capture and analysis capabilities for a variety of cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Hcs studio cell analysis software, by Thermo Fisher Scientific (2 mentions)
Glass coverslip, by Corning (2 mentions)
Bluemask 1, by ChemoMetec (2 mentions)
Xcyto 2 sample slides, by ChemoMetec (2 mentions)
Cellrox, by Thermo Fisher Scientific (1 mentions)
Antifading medium, by Vector Laboratories (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Lsm 880, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Evos xl core imaging system, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Euromedex (1 mentions)
Axio imager, by Zeiss (1 mentions)
Columbus 2, by PerkinElmer (1 mentions)
Nucblue, by Thermo Fisher Scientific (1 mentions)
Fluoroshield, by Merck Group (1 mentions)
Spotdetector bioapplication, by Thermo Fisher Scientific (1 mentions)
13 protocols using cellinsight cx7
1
Multiparametric Mitochondrial Profiling
2
Caspase-3/7 Activity Quantification in Neurons
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Caspase 3/7 positive cells were determined with CellEvent Caspase‐3/7 Green Detection Reagent following manufacturer’s instructions. Briefly, primary neurons were prepared as for LDH assays and treated with reagent diluted at 8 µM in 5% FBS NBM. Positive control wells were treated with apoptotic inducer staurosporine at 0.1 and 1 µM for 6 h. Cells were fixed in 4% (w/v) PFA and nuclei were counterstained with DAPI. Images were acquired with CellInsight CX7 (Thermo Scientific) using a 10x objective and analyzed with HCS Studio Cell Analysis Software (nuclear segmentation and casp3/7 intensities). Quantification of percentage of caspase‐3/7 positive cells was done for each well by applying a threshold manually, based on nuclear segmentation and across 81 fields.
3
Immunocytochemistry of Primary Cortical Neurons
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Primary cortical neurons grown on poly‐L‐lysine hydrobromide‐coated glass coverslips were fixed in 4% (w/v) paraformaldehyde (PFA) in 1xPBS at room temperature (RT) for 20 min, permeabilized with 1X PBS and 0.2% Triton X‐100 for 10 min at RT, and blocked for 1 h in 1x PBS and 0.1% Triton X‐100 with 5% (w/v) BSA. Neurons were incubated with primary antibodies (Appendix Table S3 ) overnight at 4°C. After three washes in 1× PBS and 0.1% Triton X‐100 for 10 min, neurons were incubated with secondary antibody anti‐rabbit and anti‐mouse (Appendix Table S3 ) for 1 h at RT, and subsequently washed three times in 1xPBS and 0.1% Triton X‐100 for 10 min. Coverslips were mounted with antifading medium (Vectashield, Vector) with DAPI and analyzed by fluorescence microscopy. Images were acquired with CellInsight CX7 (Thermo Scientific) using a 10× objective and analyzed with HCS Studio Cell Analysis Software (nuclear segmentation, NeuN and GFAP intensities). Quantification of positive cells for each of these staining was done by applying a threshold manually, based on nuclear segmentation and across 81 fields. Percentage of cells positive for NeuN and GFAP staining was quantified for each well.
4
Mitochondrial Quantification via High-Content Screening
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To reveal the changes in mitochondria quantity, we used a high-content screening system CellInsight CX7 (ThermoFisher Scientific). Cells were stained with DAPI and MitoTracker Green FM (ThermoFisher Scientific) in accordance with the manufacturer’s instructions. Every 20 min, images of the layer that is higher than the bottom were taken in the light field and fluorescence mode (excitation: 490 nm; emission: 516 nm). For each well, we analyzed 25 central fields with the total area using SpotDetector mode and measured the average fluorescence intensity caused by MitoTracker Green FM.
5
Immunocytochemistry of Induced Neurons
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The induced human neuronal cells grown on a coverslip were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min. Cells were permeabilized and blocked with PBS/0.2% Triton X-100/1% BSA for 30 min followed by incubation with the indicated primary antibodies for 2 hours at RT. After washing with PBS/1% BSA three times, cells were incubated with Alexa Fluor 647-labelled donkey anti-goat IgG, Alexa Fluor 594-labelled donkey anti-rabbit IgG or Alexa Fluor 488-labelled donkey anti-mouse secondary antibodies in the dark for 1 hour, washed with PBS/1% BSA, stained with DAPI (1 μg/ml, 10 min), and mounted on slides. Images were acquired using CellInsight CX7 (Thermo Fisher Scientific) with a 20×/0.40 NA objective (Olympus).
6
Immunofluorescence Staining of TCOF1 and rRNA
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Cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 20 min at room temperature. Cells were then permeabilised by incubating with 0.5% TritonX-100/PBS for 2 min. Glycine buffer (0.75% glycine in PBS) was used to rinse cells for 3 times. Cells were then incubated with blocking buffer (10% goat serum, 0.2% TritonX-100 in PBS) for 45 min followed by incubation with primary antibody (anti-TCOF1, 1:200 or anti-rRNA, 1:200) for 2 h and 1.5 µg/ml Alexa Fluor 647 Goat anti-Rabbit secondary antibody (Jackson ImmunoResearch #111-605-003) for 1 h. The LSM880 (Zeiss) laser scanning microscope or Thermo Scientific CellInsight CX7 High-Content Screening (HCS) Platform was used to capture images.
7
Immunocytochemistry and Histochemistry Protocols for hPSC, KER, and FIB Cells
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For immunocytochemistry, hPSC, KER, and FIB were fixed in 4% paraformaldehyde (Euromedex) and permeabilized with 0.1% Triton X-100 (Sigma, St. Louis, MI, USA). After blocking non-specific interactions with 5% bovine serum albumin (Sigma-Aldrich), samples were incubated with primary antibodies overnight at +4 °C in blocking buffer. After washing, samples were incubated 1 h with species-specific fluorophore-conjugated secondary antibodies and counterstained with DAPI at 1 μg/mL (Millipore) to allow nuclei detection. Image acquisition was performed with an inverted microscope (Axio Imager, Zeiss, Oberkochen, Germany) or the HighContentScreening module CellInsight CX7 (Thermo Fisher Scientific, Waltham, MA, USA), after cell segmentation and thresholding was based on negative control (secondary antibody only).
For immunohistochemistry and Hematoxylin Eosin staining (HE), tissues were fixed in 10% formaline (VWR) before paraffin embedding. All IHC staining was carried out on paraffin sections of 4 µm thickness using Ventana Discovery XT IHC module according to the manufacturer’s instructions. HE image acquisition was performed with EVOS™ XL Core Imaging System (Thermo Fisher Scientific). IHC image acquisition was performed with an inverted microscope (Axio Imager, Zeiss). The list of antibodies is presented inTable S2 .
For immunohistochemistry and Hematoxylin Eosin staining (HE), tissues were fixed in 10% formaline (VWR) before paraffin embedding. All IHC staining was carried out on paraffin sections of 4 µm thickness using Ventana Discovery XT IHC module according to the manufacturer’s instructions. HE image acquisition was performed with EVOS™ XL Core Imaging System (Thermo Fisher Scientific). IHC image acquisition was performed with an inverted microscope (Axio Imager, Zeiss). The list of antibodies is presented in
8
Live-Cell Imaging and Intracellular Quantification
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1 × 105 cells adhered to glass coverslips (Corning), treated with MED2-CY7 were and imaged with EVOS FL (AMG) at 10X magnification for live cell imaging. MED2-CY7 localization accomplished in 4% Paraformaldehyde-fixed, 0.1% Triton-X permeabilized cells co-stained with 1:1000 BlueMask-1(ChemoMetec) and DAPI (2μg/mL), mounted on Xcyto 2-sample slides (ChemoMetec) and imaged with Xcyto10 at 20X magnification for intracellular quantification. Mean fluorescent intensity of nuclear and cytoplasmic compartments determined using XcytoView. CellInsight CX7 (ThermoFisher) SYTOX intensity quantification acquired using a heated chamber with CO2 and imaged in 5-minute intervals at 20X magnification using Hoechst and SYTOX orange channels.
9
Automated Imaging of UPR Activation
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Immunofluorescence and direct fluorescence in SK‐N‐SH and Mif‐inducible IgM heavy chain HeLa cells and co‐culture UPR reporter assays were imaged using an automated microscopy platform (CellInsight CX7; ThermoFisher Scientific, Waltham, MA, USA). DAPI (nuclear marker) was used for autofocus and immunofluorescence experiments were imaged with a 10× objective and 25 fields of view per well and UPR reporter assays with a 20× objective and 20 fields of view per well. Image analysis was performed with Columbus 2.5 software (PerkinElmer, Groningen, The Netherlands). For both ATF4 and XBP1 immunostainings as well as UPR reporters, fluorescence intensity in the nucleus (DAPI mask) followed by background subtraction (ring around nucleus) was used as output data. BiP immunofluorescence was measured in a whole cell mask [either MAP2, Tubulin beta III isoform (SK‐N‐SH cells) or GAPDH (IgM heavy chain HeLa cells)]. Negative stainings (secondary antibody only incubation) were also used to determine background values for immunofluorescence experiments and values were subtracted from output data of ATF4, XBP1, and BiP analyses. In experiments with SK‐N‐SH cells, the highest and lowest data points (one pair/experiment) were excluded from the datasets. Data of UPR reporter experiments were normalized by rescaling between 1 (minimum; EtOH/Mif 48 h) and 10 (maximum; TG) per experiment.
10
Live-Cell Imaging and Intracellular Quantification
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