Mitochondria-specific Mito Tracker (Red CMX Ros) fluorescent dye was used to monitor the variation in mitochondrial membrane potential (MMP) as described by Farooqui et al., 2017 [30 (link)]. In short, cells (seeded in a 12-well plate) were treated with several doses of rutin (80–320 μg/ml). After 12 h of treatment, HeLa cancer cells were washed with phosphate buffer saline (PBS; twice) and then exposed to 3.5% of paraformaldehyde (for cell fixation process) at 37°C for 15 min. Finally, treated cells were stained with Mito Tracker Red (25 μg/ml) dye to observe changes in MMP under fluorescence microscope (FLoid Cell Imaging Station, Thermo Fisher Scientific, U.S.A.).
Floid cell imaging station
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, India, Australia, Canada
The FLoid Cell Imaging Station is a compact and easy-to-use fluorescence microscope designed for cell imaging. It allows users to capture high-quality images and videos of cells, tissues, and other biological samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (9 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (4 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fluoro carbonyl cyanide phenylhydrazone (fccp), by Cayman Chemical (3 mentions)
Jc 1 dye, by Adipogen (3 mentions)
Rhodamine 123, by BD (3 mentions)
Deepred dye, by Thermo Fisher Scientific (2 mentions)
Prolong anti fade reagent with dapi, by Thermo Fisher Scientific (2 mentions)
Countess 2 automated cell counter, by Thermo Fisher Scientific (2 mentions)
Lsr fortessa flow cytometer with high throughput sampler, by BD (2 mentions)
Mitotracker deep red fm, by Thermo Fisher Scientific (2 mentions)
Chromium controller, by 10x Genomics (2 mentions)
Influx cell sorter, by BD (2 mentions)
Phycoerytherin pe anti human cd14 antibody, by BD (2 mentions)
Dmi6000b microscope, by Leica (2 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (2 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (2 mentions)
173 protocols using floid cell imaging station
1
Mitochondrial Membrane Potential Assay
2
Nematode Identification in Ladybirds
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Adult beetles and L4 larvae from the greenhouse-reared population (n = 80 each), as well as laboratory-reared beetles (n = 142) and individuals collected around Ober-Mörlen in late October 2017 (n = 324) were dissected immediately after sampling. Nematodes from infected ladybirds were collected in PBS and the species identified using a FLoid® Cell Imaging Station (Thermo Fisher Scientific) to capture images at different life stages. The nematodes were centrifuged at 16,000 × g for 15 min at 4 °C and the pellet was resuspended in nuclease-free water before lysis and amplification of the 18S SSU rRNA gene as previously described50 (link) using GoTaq® G2 DNA Polymerase (Promega). The PCR products were purified and transferred to the vector pGEM®-T Easy (Promega) for sequencing, followed by sequence analysis using Geneious v10.2.251 (link). A consensus sequence was generated by the de novo assembly of the three overlapping sequence fragments and a BLAST search against the NCBI nr/nt database was carried out using Megablast with standard parameters.
3
Quantifying Oxidative Stress and Mitochondrial Function
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HUVECs/MDMs were stimulated with 80 μg/mL oxLDL and/or 40 μM CORM-A1 for 24 h. Control and treated cells were stained with 10 μM of 2′, 7′-dichlorofluorescein (H2DCFDA; for intracellular oxidative stress)/50 nM MitoTracker with 1 μg/mL Hoechst 33,342 (for mitochondrial mass) or with JC-1 (5 μg/mL; for mitochondrial membrane potential). Stains were added to control and treated cells in fresh incomplete media for 15 min/30 min, respectively, at 37 °C, washed with 1X PBS and photographed on a FLoid Cell imaging station (Thermo Fisher Scientific, USA).
4
Imaging Apoptotic Thymocyte Uptake by Macrophages
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Apoptotic thymocytes were stained with 2.5 µM DeepRed dye (Invitrogen, Carlsbad, CA, USA) for 24 h according to the protocol provided by the manufacturer, while BMDMs were stained with 10μM carboxyfluorescein diacetate succinimidyl ester (CFDA-SE; Thermo Fisher Scientific, Waltham, MA, USA). Apoptotic thymocytes were added to 2 × 105 C57BL/6 macrophages in 1:5 macrophage:target cell ratio for 1 h, then the remaining cells were washed away. BMDMs were then fixed with 1% paraformaldehyde. Pictures were then taken on a fluorescent microscope (FLoid™ Cell Imaging Station, ThermoFisher, Waltham, MA, USA.
5
Quantification of Lipid Accumulation in HUVEC
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HUVEC were seeded to detect lipids as well as to perform the MTT assay under the same experimental conditions. Oil Red O Staining was used to detect neutral lipids. After the 24h treatments, the cells were washed three times with PBS, fixed in PFA 10% for 30 min at room temperature, washed once again with PBS, and then stained with 60% filtered Oil Red O stock solution (Sigma-Aldrich) for 20 min. After extensive washing, the Oil Red O was solubilized in 100% isopropanol, was quantified by measuring the absorbance at 500 nm, and was normalized to the cell number by MTT assay [24 (link),25 (link)] after image acquisition using FLoid Cell Imaging Station (Thermo Fisher Scientific). To further confirm the results, staining with BODIPY 493/503 was performed (see dataverse at the following link https://dataverse.unimi.it/dataverse/biomedicines/ accessed on 30 September 2021). The results are the mean of three independent experiments performed in triplicate ± SD.
6
3D Tumoroid Formation and Quantification
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Tumoroids were formed in the 3D culture systems using NanoCulture Plate (NCP) (Medical & Biological Laboratories, Nagoya, Japan) or ultra-low attachment (ULA) culture plates/dishes (Greiner, Kremsmunster, Austria) within mTeSR1 stem-cell medium (Stemcell Technologies, Vancouver, BC, Canada) or the above-mentioned serum-containing medium as described previously [34 (link),35 (link),36 (link),42 (link)].
For quantification of tumoroids size and number, cells were seeded in a 96 well NCP for 14 days at a concentration of 5.0 × 103 cells in 200 μL mTeSR1 or RPMI-1640 media with 10% FBS. Tumoroid maturation was monitored every day and photographed using the Floid cell imaging station (Thermo Fisher, Waltham, MA, USA) from day 1 until day 7 and a BZ-X microscope (Keyence, Osaka, Japan) starting from day 10 until the end of the experiment day 14. The tumoroid size was measured using Image J software (NIH, Bethesda, MD, USA).
For quantification of tumoroids size and number, cells were seeded in a 96 well NCP for 14 days at a concentration of 5.0 × 103 cells in 200 μL mTeSR1 or RPMI-1640 media with 10% FBS. Tumoroid maturation was monitored every day and photographed using the Floid cell imaging station (Thermo Fisher, Waltham, MA, USA) from day 1 until day 7 and a BZ-X microscope (Keyence, Osaka, Japan) starting from day 10 until the end of the experiment day 14. The tumoroid size was measured using Image J software (NIH, Bethesda, MD, USA).
7
Serological Assays for Arbovirus Exposure
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To determine the seroconversion of animals, all JEV, YFV, and ZIKV IgG indirect immunofluorescence assays (IIFAs) were performed as per the manufacturer’s instructions (Euroimmun, Lübeck, Germany), except for the use of labeled secondary antibody and the mounting agent glycerine, which were replaced by Alexa Fluor 488 goat anti-mouse IgG (A-11029; Thermo Fisher Scientific) and 4′,6-diamidino-2-phenylindole (DAPI; ProLong antifade reagent with DAPI; Thermo Fisher Scientific), respectively. Serum from nonvaccinated animals served as a naive, negative control. Slides were visualized using a fluorescence microscope (FLoid cell imaging station; Thermo Fisher Scientific).
8
Quantitative Live/Dead Viability Assay
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Cell death was determined by Live/Dead viability assay, as described previously (Doria et al., 2013 (link)). Briefly, neurons were stained with 2 μM calcein acetoxymethyl ester (AM) and 2 μM ethidium homodimer‐1 for 15 min and the fractions of live (calcein AM positive) and dead (ethidium homodimer-1 positive) cells were determined by microscopy. Neurons were visualized by fluorescence microscopy FLoid® Cell Imaging Station (Thermo Scientific, Waltham, MA, USA) and scored by a blinded observer. A minimum of 300 cells were analyzed per well in triplicates using the ImageJ™ software. Dead cells were expressed as a percentage of the total number of cells.
9
Autophagy Modulation and ATXN1 Aggregation
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Daoy 30Q-RFP cells or Daoy 82Q-RFP cells were grown in culture and plated onto Nunc Lab Tek II 8-well chamber slides (ThermoFisher Scientific 154,534; Waltham, MA). The state of ATXN1 aggregation during the induction and inhibition of autophagy was determined using an autophagy assay kit (AbCam ab139484; Cambridge, MA). Briefly, 18 h following plating, cells were treated for 24 h with 500 nM rapamycin, dissolved in dimethylsulfoxide (DMSO; Sigma-Aldrich D2650;St. Louis, MO), and 60 µM chloroquine, dissolved in distilled deionized water, DMSO vehicle control or left untreated. Cells were stained for autophagy following the kit instructions and imaged on a FLoid Cell Imaging Station (ThermoFisher Scientific). Counts were performed from two randomly selected non-overlapping fields per chamber from each of three independent experiments.
10
Live/Dead Cell Viability Assay
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The live/dead assay was used to visually differentiate between live and dead cells, and
to assess morphological changes that occurred following treatment with the local
anesthetics. Chondrocytes were plated in 8-well chamber slides (Iwaki, Tokyo, Japan) at a
density of 2.0 × 104 cells per well for 24 hr then treated with 10-DMEM as the
control or with local anesthetics in 10-DMEM as described above. After treatment, the
live/dead reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to the culture
wells and incubated for 30 min at 37°C and 5% CO2, then the solution was
carefully removed and replaced with PBS for viewing. A FLoid Cell Imaging Station (Thermo
Fisher Scientific) was used for fluorescent microscopy and images were obtained under
green (FITC filter) and red (Texas red filter) light. Where necessary, photo contrast and
brightness were adjusted using ImageJ software (National Institute of Health, Bethesda,
MD, USA). The live/dead assay analyses cell membrane integrity and permeability by
differential staining with either calcein for live cells or ethidium homodimer (EthD-1)
for dead cells. Calcein is well retained within live cells, producing a uniform green
fluorescence whereas EthD-1 enters cells with damaged membranes and produces bright red
fluorescence.
to assess morphological changes that occurred following treatment with the local
anesthetics. Chondrocytes were plated in 8-well chamber slides (Iwaki, Tokyo, Japan) at a
density of 2.0 × 104 cells per well for 24 hr then treated with 10-DMEM as the
control or with local anesthetics in 10-DMEM as described above. After treatment, the
live/dead reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to the culture
wells and incubated for 30 min at 37°C and 5% CO2, then the solution was
carefully removed and replaced with PBS for viewing. A FLoid Cell Imaging Station (Thermo
Fisher Scientific) was used for fluorescent microscopy and images were obtained under
green (FITC filter) and red (Texas red filter) light. Where necessary, photo contrast and
brightness were adjusted using ImageJ software (National Institute of Health, Bethesda,
MD, USA). The live/dead assay analyses cell membrane integrity and permeability by
differential staining with either calcein for live cells or ethidium homodimer (EthD-1)
for dead cells. Calcein is well retained within live cells, producing a uniform green
fluorescence whereas EthD-1 enters cells with damaged membranes and produces bright red
fluorescence.
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