BJ cells (106 cells) were fixed using 4% solution of p-formaldehyde in PBS containing 0.01% Triton X-100 at room temperature for 10 min, washed using PBS and suspended in 70% ethanol at 4 °C for 20 min. To avoid unspecific binding, fixed cells were pre-incubated with 1% BSA in PBS for 20 min and then incubated with primary antibodies anti-p53 (1:200, MA5-12557) and anti-p21 (1:200, MA5-14949) at room temperature for 1 h and secondary antibody conjugated to Alexa Fluor Plus 488 (1:1000, A32723, A32731) (Thermo Fisher Scientific, Warsaw, Poland) at room temperature for 1 h. Fixed cells were then stained using 5 μg/ml PI solution for 10 min. Digital cell images were captured and intracellular localization of p21 and p53 was analyzed using flow imaging cytometry (Amnis® FlowSight® imaging flow cytometer and IDEAS software version 6.2.187.0, Merck Millipore, Warsaw, Poland).
Ideas software version 6
Manufactured by Merck Group
Sourced in United States, Germany
IDEAS software version 6.2 is a lab equipment product by Merck Group. It is a software application that provides data analysis and visualization tools for scientific researchers. The software core function is to facilitate the processing and interpretation of experimental data.
Automatically generated - may contain errors
Lab products found in correlation
Flowsight imaging flow cytometer, by Merck Group (3 mentions)
Amnis flowsight imaging flow cytometer, by Merck Group (2 mentions)
Alexa fluor plus 488, by Thermo Fisher Scientific (1 mentions)
Dp72 ccd camera, by Olympus (1 mentions)
Sybr green 1 solution, by Thermo Fisher Scientific (1 mentions)
Facscalibur, by BD (1 mentions)
Cd206 alexa flour 488 clone 19, by Thermo Fisher Scientific (1 mentions)
Prism 6, by GraphPad (1 mentions)
9 protocols using ideas software version 6
1
p21 and p53 Expression Analysis in BJ Cells
2
Microalgal Cell Size Analysis
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The size of microalgal cells was analyzed using an Olympus BX61 differential interference contrast microscope equipped with a DP72 CCD camera and Olympus CellF (Olympus, Warsaw, Poland). Moreover, the subpopulations of cells in terms of their cell size and formation of cell aggregates were investigated using Amnis® FlowSight® imaging flow cytometer and IDEAS software version 6.2.187.0 (Merck Millipore, Warsaw, Poland). 10,000 events were analyzed for each sample triplicate using two channels (bright field), namely Ch01 (435–480 nm) and Ch09 (570–595 nm) with the 488 nm and 642 nm lasers. Five subpopulations of cells were considered, namely single cells sized 5–10 µm (R1), single cells sized 1–5 µm (R2), large single cells sized 10–15 µm and autosporangia (R3), cell aggregates sized over 15 µm (R4) and dividing cells with autospores (R5). Representative dot plots and cell images are presented.
3
Cell Cycle Analysis via Flow Cytometry
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Cells were treated with 1 mg/mL RNAse A at 37°C for 1 h followed by 5 mg/mL Proteinase K at 37°C for another 1 h. After washing with TE buffer, cells were resuspended in a SYBR Green I solution (Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. Flow cytometry (FACSCalibur; BD Biosciences, San Jose, CA, USA) and the IDEAS software version 6.2.187.0 (Merck KGaA, Darmstadt, Germany) were used to determine the phases of the cell cycle.
4
Quantifying RL2 Internalization and Cell Death
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Analysis of RL2 internalization and cell death induction was performed with FlowSight® Imaging Flow Cytometer (Amnis/Merck Millipore, Darmstadt, Germany). MDA-MB-231 or MCF-7 cells were treated with RL2 or Rhodamine-labelled RL2 [8 (link)] for measuring cell death or internalization, respectively. Samples for measuring cell death were additionally stained with propidium iodide (PI). The samples were excited with a 488 nm laser. Emission was detected in channel 4 and bright field images were acquired in channels 1 and 9. For every sample, 10,000 events were recorded. Data were analyzed with IDEAS software version 6.2 (Amnis/Merck Millipore, Darmstadt, Germany). For internalization, Rhodamine-positive cells were taken as RL2-positive cells. RL2-induced cell death was calculated via the percentage of PI positive cells.
5
Imaging Flow Cytometry for Extracellular Vesicle Analysis
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ImageStream combines flow cytometry with fluorescence imaging technology and can resolve much smaller particles. The tetraspanin marker, CD63 eFlour450 (clone H5C6; Affymetrix, Inc., Santa Clara) was used as a positive marker to distinguish exosomes from other types of EVs and calibration beads [32] (link), [43] (link). The following antibodies were used to characterize EVs: HLA-DR APC (clone LN3; Affymetrix, Inc., Santa Clara), CD9 PE (clone M-L13; BD Biosciences, San Jose, CA), CD81 PE-Cy7 (clone 5A6; BioLegend, Inc., San Diego, CA), Grp94 DyLight 488 (clone 9G10; Enzo Life Sciences, Inc., Framingdale, NY), and ARF6 APC (AssayPro, LLC, St. Charles, MO) [43] (link), [44] (link), [45] , [46] (link). The stained samples were imaged at 60 × magnification with extended depth of field (EDF), while acquiring data on channels Ch01, Ch03, Ch06, Ch07, Ch09, Ch11 and Ch12. For acquisition of MitoTracker Green, MitoTracker Red, MitoSox, or CellLight Mitochondrial GFP, channel Ch02 was used for green fluorescence and Ch03 for red. Appropriate controls, single color stains, and calibration beads were used to adjust spectral compensation. A total of 5000 events were acquired. Channels Ch01 and Ch09 were used as brightfields, and Ch12 was used for side-scatter. The acquired data was analyzed using IDEAS software version 6.2 (EMD Millipore, Bellerica, MA).
6
Apoptosis Analysis of MDA-MB-231 Cells
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For apoptosis analysis, MDA-MB-231 cells (1 × 105/well) were cultured in 6-well for 24 h and treated with AITC at 10 µM. After 24 h of treatment, floating and adherent cells were collected and stained with Annexin V-FITC and PI following the manufacturer’s instructions. Stained cells were then analyzed using an imaging flow cytometer named Flow Sight (Amnis Corporation, Seattle, WA, USA) and IDEAS Software version 6.2 (EMD Millipore, Burlington, MA, USA).
7
Multiparameter Macrophage Phenotyping
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Macrophages were stained with CD64 Percp-cy5 (clone 10.1, BD Pharmingen), CD206 Alexa Flour 488 (clone 19.2, eBioscience), and CD163 Pecy7 (clone eBioGHI/61, Thermo Fisher Scientific) for ImageStream analyses. For Image Stream, samples were imaged at 60× magnification while acquiring data on different channels. The machine was calibrated using single color stained and unstained controls, as well as calibration beads to adjust the noise levels and spectral compensation. Brightfield data were collected on channels Ch01 and Ch09, and side-scatter was collected on Ch12. 5000 events were acquired and used for analysis in IDEAS software version 6.2 (EMD Millipore, Billerica, MA, USA).
8
Flow Cytometry Analysis of MSN Nanoparticle Uptake
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Flow cytometry was utilized when the uptake of MSN nanoparticles by cancer cells was examined. In these cases, 3×105 MCF-7 or MCF-7 KCR cells were seeded into 6-well plates in complete culture media (RPMI medium complemented with 10% FBS, 2 mM L-glutamine, 0.01% streptomycin and 0.005% penicillium). On the following day, cells were treated with 0.1 mM RhoB@MSNs in the above described complete culture media for 24 hours. Then, the RhoB@MSN-containing medium was removed, samples were washed with PBS, and cells were incubated in serum-free media for 24 or 48 hours. As a reference, in independent samples, cells were treated only for 24 hours in full culture media containing 0.1 mM RhoB@MSNs. Before flow cytometry analysis, all samples were washed with PBS, trypsinized, centrifuged (400 g, 5 min), resuspended in 200 µL PBS and measured by FlowSight Imaging Flow Cytometer (Amnis, EMD Millipore, Burlington, MA, USA). Data were analyzed by Ideas Software Version 6.2 (Amnis, EMD Millipore, Burlington, MA, USA). Statistical analysis was performed in GraphPad Prism 6 software using two-way ANOVA Sidak’s multiple comparisons test.
9
Imaging Flow Cytometry Analysis of Cell Death
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Analysis of cell death induction was performed with FlowSight® Imaging Flow Cytometer (Amnis/MerckMillipore, USA). MDA-MB-231 cells were treated with RL2. Samples were stained with annexin V-fluorescein isothiocyante and propidium iodide. Data were analyzed with IDEAS software version 6.2 (Amnis/MerckMillipore, Darmstadt, Germany), as described previously (Pietkiewicz et al., 2015 (link)).
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