Purified parasite stages harvested 4 dpt were stained with 5 μg/mL DAPI (4′,6-diamidino-2-phenylindol) working solution, washed with PBS and transferred to 1 mL of PBS solution to be immediately processed. The different parasite stages were subjected to fluorescence analysis in an Amnis® FlowSight® imaging flow cytometer (Luminex, Austin, USA) and were evaluated by size, shape and fluorescence intensity. A total of 10 000 recordings were measured for every replicate (n = 5) and gated with software IDEAS® 6.2.64.0 (Luminex) based on size, which we used for previous morphological studies, and intensity, which made it possible to differentiate debris from different parasite cells.
Amnis flowsight imaging flow cytometer
Manufactured by DiaSorin
Sourced in United States
The Amnis FlowSight Imaging Flow Cytometer is a laboratory instrument designed for high-resolution image capture and analysis of individual cells in flow. It combines the capabilities of flow cytometry with advanced imaging technology to provide detailed visual information about cell morphology and intracellular features.
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Lab products found in correlation
Pe anti human asc clone hasc 71, by BioLegend (1 mentions)
Muse ki67 proliferation kit, by DiaSorin (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
Cyto fast fix perm buffer, by BioLegend (1 mentions)
Amnis flowsight, by DiaSorin (1 mentions)
Cell staining buffer, by BioLegend (1 mentions)
Cellmask green actin tracking stain, by Thermo Fisher Scientific (1 mentions)
Prism 9, by GraphPad (1 mentions)
Lc3b antibody, by Cell Signaling Technology (1 mentions)
A32723, by Thermo Fisher Scientific (1 mentions)
12 protocols using amnis flowsight imaging flow cytometer
1
Parasite Stage Identification via Flow Cytometry
2
Quantitative Analysis of ASC in PBMCs
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PBMCs (1 × 106), stimulated as described above, were fixed with 100 μL of Paraformaldehyde (PFA) (1%) (BDH, United Kingdom), permeabilized with 100 μL of saponin (0.1%) (Life Science VWR, Lutterworth, Leicestershire, LE), and stained with PE-antihuman ASC (clone HASC-71, isotype mouse IgG1, Biolegend, San Diego, CA, United States) for 1 h at room temperature; cells were then washed with PBS, centrifuged at 1,500 rpm for 10 min, resuspended in 50 μL of PBS, and examined using the AMNIS FlowSight Imaging Flow Cytometer (Luminex Corporation Austin, TX). Results were analyzed using an analysis software (IDEAS). The IDEAS image analysis software allows quantification of cellular morphology and fluorescence at different cellular localizations by defining specific cellular regions (masks) and mathematical expressions that uses image pixel data or masks (features).
3
Quantifying Proliferation and Apoptosis
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As a marker of cell proliferation, Ki67 immunostaining was considered. Muse™ Ki67 Proliferation Kit was used (MCH100114, Luminex Corporation, Austin, TX, USA). Apoptosis-associated DNA strand breaks were revealed using TUNEL (TdT-mediated dUTP-X nick end labeling) assay (11684795910, In Situ Cell Death Detection Kit, Fluorescein, Roche, Basel, Switzerland). Digital cell images were captured and Ki67-positive cell population and DNA damage-positive cell population were analyzed using an Amnis® FlowSight® Imaging Flow Cytometer and IDEAS software version 6.2.187.0 (Luminex Corporation, Austin, TX, USA). Representative histograms are presented.
4
Cell Cycle Analysis by Imaging Flow Cytometry
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For DNA content-based analysis of the cell cycle, imaging flow cytometry was used. After 5-azaC post-treatment, cells were fixed using 4% paraformaldehyde (PFA) at room temperature for 20 min, washed in DPBS, resuspended in 70% ethanol, and incubated on ice for 20 min. DNA was visualized using propidium iodide (PI) staining. The subpopulations of cells at the phases G0/G1, S, and G2/M of the cell cycle were analyzed using Amnis® FlowSight® imaging flow cytometer and IDEAS software version 6.2.187.0 (Luminex Corporation, Austin, TX, USA).
5
Imaging Flow Cytometry of NF-κB Activation
6
Cell Cycle and Apoptosis Analysis
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Cell cycle and apoptosis analysis was done using propidium iodide staining on Amnis® FlowSight imaging flow cytometer (Luminex Corp, Austin, TX, USA). Briefly, MSTO-211H cells were seeded in TC-treated 6-well plates (500,000 cells/well, n = 3) and were incubated overnight for attachment. The following day, cells were treated with QA (2.5- and 5-µM) for 24 h. After 24 h, treatments were removed, and cells were washed thoroughly with 1X PBS, followed by trypsinization for cell harvesting. Cell pellets were collected and fixed with 70% ice-cold ethanol for 1 h. Following fixing, cells were washed with 1X PBS and incubated with RNA-ase (100 µg/mL) and propidium Iodide (10 µg/mL) for 30 min in dark. Cells were then analyzed (50,000 counts/sample) using Amnis® Flowsight® (Luminex Corp., Austin, TX, USA). Data were analyzed using IDEAS® software ver. 6.2 (Luminex Corp., Austin, TX, USA).
7
Actin Dynamics Quantification Protocol
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Throughout the 10 cm dish plates, 5 × 105 cells were spread evenly and left overnight. After incubation for 24 h in the presence of appropriate enzymes, genistein, or its vehicle, CellMask™ Green Actin Tracking Stain (Thermo Scientific, Waltham, MA, USA), was added according to the manufacturer’s instructions. Samples were collected through trypsinization and centrifugation processes (4 washes in cell staining buffer (BioLegend, San Diego, CA, USA) and analyzed and quantified via an Amnis FlowSight Imaging Flow Cytometer, Luminex (Austin, TX, USA), using IDEAS 6.2 Software. After normalizing the obtained data, the relevant graphs of relative fluorescence intensity were prepared and statistical analysis (ANOVA) was performed (GraphPad Prism 9).
8
Flow Cytometric Analysis of Cultured Cells
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The flow cytometric measurements were performed on the Amnis® FlowSight® Imaging Flow Cytometer (Luminex, Austin, TX, USA) kindly provided by the Moscow State University Development Program PNR5. The Amnis® FlowSight® Imaging Flow Cytometer was equipped with a 488 nm laser (60 mW) and a SSC laser (10 mW). Cells were removed from the culture vessel surface into suspension with trypsin/versene (1:1) 5 min at 3000× g, +4 °C and resuspended in 50 µL of PBS. For all probes, the integrated intensity of each cell was measured. Flow cytometric data analysis was performed with the IDEAS software ver. 6.3 (Luminex, Austin, TX, USA).
9
Mitochondrial Membrane Potential Assay
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Mitochondrial membrane potential changes
in MRC5-SV40 cells were assessed by the fluorescence of tetramethylrhodamine
methyl ester (TMRM) (Invitrogen) or JC-1 (Lumiprobe),71 (link) using an Amnis FlowSight Imaging Flow Cytometer (Luminex
Corporation, Seattle, WA). With 100 nM TMRM, the cells were stained
for 15 min. Then, the cells were incubated with SF-Cn-TPP or SF6847 at various concentrations for 1 h. After
this treatment, the cells were detached with trypsin/versene and washed
from the medium with phosphate-buffered saline. The median fluorescence
of the cells was analyzed with a flow cytometer (excitation 548 nm,
emission 574 nm). With 1 μg/mL JC-1, the cells were incubated
at 37 °C for 30 min, then washed from the medium, and then analyzed
with the flow cytometer. Membrane potential was assessed by the ratio
of the fluorescence in the red channel (exc. 514 nm, em. 590 nm),
emitted by the so-called “J-aggregates” in highly energized
mitochondria, and the fluorescence in the green channel (exc. 514
nm, em. 529 nm), emitted by JC-1 in de-energized mitochondria.
in MRC5-SV40 cells were assessed by the fluorescence of tetramethylrhodamine
methyl ester (TMRM) (Invitrogen) or JC-1 (Lumiprobe),71 (link) using an Amnis FlowSight Imaging Flow Cytometer (Luminex
Corporation, Seattle, WA). With 100 nM TMRM, the cells were stained
for 15 min. Then, the cells were incubated with SF-Cn-TPP or SF6847 at various concentrations for 1 h. After
this treatment, the cells were detached with trypsin/versene and washed
from the medium with phosphate-buffered saline. The median fluorescence
of the cells was analyzed with a flow cytometer (excitation 548 nm,
emission 574 nm). With 1 μg/mL JC-1, the cells were incubated
at 37 °C for 30 min, then washed from the medium, and then analyzed
with the flow cytometer. Membrane potential was assessed by the ratio
of the fluorescence in the red channel (exc. 514 nm, em. 590 nm),
emitted by the so-called “J-aggregates” in highly energized
mitochondria, and the fluorescence in the green channel (exc. 514
nm, em. 529 nm), emitted by JC-1 in de-energized mitochondria.
10
Oxidative Stress Monitoring in Cell Lines
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MRC5-SV40 or primary human fibroblasts cell lines were incubated with erastin (10 mM) or BSO (1 mM) for 17 h, and then cells were stained with 100 nM MitoCLox (1 h) or 1.8 μM DCFH2 DA (30 min). After this, cells were stripped with trypsin/versene, and the medium containing cells was centrifuged in 1.5 mL tubes (1000 r min−1, 3 min) at 4 °C. The centrifuged cells were redispersed in PBS (0.05 mL). Each sample was measured until 2000 events had been collected. Flow cytometry analyses were performed using an Amnis FlowSight Imaging Flow Cytometer (Luminex Corporation, Seattle, WA, USA) with excitation at 488 nm and the detection channels 505–560 (Ch2) and 595–642 (Ch4). For ratiometric analysis, the Amnis IDEAS® 6.2 (Luminex, Seattle, WA, USA) image analysis software was used.
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