The assessment of ΔΨm was performed according to Ref. [95 (link)]. Briefly, cells were incubated for 20 min at RT in the dark, with a deep-red MitoTracker (20 nM final concentration) compound (cat #M22426, Thermo Scientific, Waltham, MA, USA). Cells were analyzed using fluorescence microscopy Floid Cells Imaging Station microscope (cat# 4471136, Life Technologies, Carlsbad, CA, USA), or a BD LSRFortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was conducted 3 times, and 10,000 events were acquired for analysis. MitoTracker highly positive cells were selected located between 104 and 106. No discrimination by complexity was made. Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software (BD Biosciences, Franklin Lakes, NJ, USA).
Lsrfortessa 2 flow cytometer
Manufactured by BD
Sourced in United States
The BD LSRFortessa II is a flow cytometer designed for high-performance multiparameter analysis. It features a compact design, multiple laser configurations, and advanced optics to enable the detection and analysis of a wide range of cellular properties and characteristics.
Automatically generated - may contain errors
Lab products found in correlation
Floid cells imaging station microscope, by Thermo Fisher Scientific (2 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (2 mentions)
Mitotracker, by Thermo Fisher Scientific (1 mentions)
Fitc anti mouse cd45 antibody, by BioLegend (1 mentions)
Pe anti mouse f4 80 antibody, by BioLegend (1 mentions)
Lysotracker green dnd 26, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Cd45 percp cy5, by BD (1 mentions)
Caspase 3, by Merck Group (1 mentions)
Anti puma, by Abcam (1 mentions)
Bodipy fl c16, by Thermo Fisher Scientific (1 mentions)
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
Cellrox deep red reagent, by Thermo Fisher Scientific (1 mentions)
Zombie aqua fixable viability dye, by BioLegend (1 mentions)
Cytofix solution, by BD (1 mentions)
Perm wash solution, by BD (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Rabbit anti caspase 3, by Merck Group (1 mentions)
15 protocols using lsrfortessa 2 flow cytometer
1
MitoTracker Assay for Mitochondrial Membrane Potential
2
Mitochondrial Membrane Potential Analysis
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Assessment of the ΔΨm was performed according to Ref. [36 (link)]. We incubated cells (1x105) for 20 min at r. t. in the dark with a deep red mitotracker (20 nM final concentration) compound (Thermo Scientific, cat# M22426). Cells were analyzed by using a BD LSRFortessa II flow cytometer (BD Biosciences). The experiment was conducted three times, and 10,000 events were acquired for analysis. Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software.
3
Detecting Leukemic Cells in Mice
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Ba/F3 P210 BCR-ABL cells (5 × 106 cells) were administered via intravenous (i.v.) injections into the lateral tail veins of immunosuppressed mice (n = 12), on days 11, 14, and 17 (Fig. 1 ), thereafter called leukemia mice (n = 12). To determine the presence of Ba/F3 BCR-ABL P210 cells in mice peripheral blood, we used the presence of both CD45 (a leukocyte marker, most expressed in lymphocytes) and F4/80 surface (https://cell.brc.riken.jp/en/rcb/baf3 ; Last update: 2022.07.25), and ABL proteins according to supplier’s recommendation. Briefly, total blood was collected, and 30 μl of the sample was incubated with rat anti-mouse FITC-CD45 (FITC anti-mouse CD45 Antibody, Cat# 103108, Biolegend), rat anti-mouse PE-F4/80 (PE anti-mouse F4/80 Antibody, Cat# 123110, BioLegend), and anti-ABL (c-Abl Antibody; Cat# MA5-14398; Invitrogen) antibodies for 15 min. Then, samples were incubated in red blood cell lysis solution for 30 min at 37 °C. Finally, the sample was centrifuged at 2000 rpm for 10 min. and incubated with DyLight 488 donkey anti-mouse antibody (1:500) (to identify ABL primary antibody), rinsed and resuspended for analysis on a BD LSR Fortessa II flow cytometer (BD Biosciences). Fifty thousand events were acquired, and the acquisition analysis was performed using FlowJo 7.6.2 Data Analysis Software.
4
Quantifying Lysosomal Complexity in Cells
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To analyze lysosomal complexity, cells were incubated with the cell-permeable, non-fixable, green, fluorescent dye LysoTracker Green DND-26 (50 nM, cat #L7526, Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 37 °C. Cells were then washed, and LysoTracker fluorescence was determined by analysis of fluorescence microscopy images in a Floid Cells Imaging Station microscope (Cat# 4471136, Life Technologies, Carlsbad, CA, USA), or flow cytometry using a BD LSRFortessa II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). The experiment was conducted 3 times, and 10,000 events were acquired for analysis. Flow cytometry analysis for LysoTracker/SSCA was performed by selecting, in the FL-1 channel, all cells with LysoTracker reactivity (>99%), in order to perform the analysis of the total LysoTracker-positive population. SSCA parameter was adjusted to the mean fluorescence of the control (UNT; 40 K ± 3.5 K) plus two standard deviations (i.e., values above 47 K). Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software (BD Biosciences, Franklin Lakes, NJ, USA).
5
Detailed Flow Cytometry Procedure for Bone Marrow Analysis
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For bone marrow analysis, cells were gathered and treated with red blood cell lysis buffer (Biogems, 64010-00) to lyse red blood cells. Single cells suspensions were obtained through filtration with a 40-μm cell strainer. Thus, the composition of tdTomato+ cells was assessed. Using Hoechst 33342 (Invitrogen, H3570, 1:1000), isolated cells were stained. As directed by the manufacturer, fluorochrome-conjugated antibodies (1 μg per 106 cells) were used to label the cells extracted from mice cardiac allografts. Conjugated-antibodies used include, CD45-percp-cy5.5 (BD pharmingen, 550994, 1:100) for 30 min. Then, to exclude dead cells, the stained cells were rinsed in PBS before being dyed for 20 minutes with LIVE/DEAD ™ Fixable Near-IR Dead Cell Stain Kit (Invitrogen, L34975 1:1000). Afterwards, cells were washed with PBS and re-suspended with PBS containing 5% BSA. Using a CytoFLEX LX flow cytometry equipment, fluorescence-tagged cells were evaluated, and data were analyzed by CytExpert in accordance with protocols. The cell sorting process was performed using BD LSR Fortessa II flow cytometer (BD Biosciences), and FlowJo v10 software (BD Biosciences, USA) was used for subsequent analysis.
6
Quantifying Apoptosis in Cell Lines
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After each treatment with or without TPGS, cells (1 × 105 cells/well) were fixed in 80% ethanol and stored at −20° C overnight. Then, cells were washed with PBS and permeabilized with 0.2% Triton X-100 plus 1.5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) for 30 min. Cells were washed and incubated with anti-PUMA (Abcam, cat. No. ab-9643) and caspase-3 (Rabbit, Millipore, cat. No. AB3623) primary antibodies (1 : 500, diluted in PBS containing 0.1% BSA). Subsequently, the cells were washed and incubated with (1 : 500) Dylight donkey anti-rabbit (594 nm, cat. No. DI-1094) or -mouse (488 nm, cat. No. DI-2488) secondary antibodies for 30 min at RT in the dark. After washing with PBS, the cells were suspended in 500 μL of PBS. The analysis was performed on a BD LSRFortessa II flow cytometer (BD Biosciences). Cells without primary antibodies served as a negative control. For assessment, it was acquired 10000 events and quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software.
7
Flow Cytometry-Based Cell Viability Assay
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Cell viability was assessed using the zombie aqua fixable viability dye (Biolegend, San Diego, California, USA). PBMCs were than either barcoded (procedure described later) or immediately stained following the manufacturers’ recommendations. For intracellular staining cells were fixed with BD Cytofix solution and washed in BD Perm/Wash solution (BD Bioscience).
The dyes 6-NBDG, Bodipy FLC16, CellROX Deep Red Reagent, MitoSox Red Mitochondrial Superoxide Indicator, MitoTracker Green FM (ThermoFisher Scientific), and TMRE (Cayman Chemical, Ann Arbor, Michigan) were used according to manufacturers’ instructions.
Samples were measured with a BD FACSCanto II or BD LSR Fortessa II flow cytometer using the BD FACSDiva Software. Data was analysed with the FlowJo V10 software (BD Bioscience).
A list of all antibodies and dyes can be found in Supplementary TableS4 online.
The dyes 6-NBDG, Bodipy FLC16, CellROX Deep Red Reagent, MitoSox Red Mitochondrial Superoxide Indicator, MitoTracker Green FM (ThermoFisher Scientific), and TMRE (Cayman Chemical, Ann Arbor, Michigan) were used according to manufacturers’ instructions.
Samples were measured with a BD FACSCanto II or BD LSR Fortessa II flow cytometer using the BD FACSDiva Software. Data was analysed with the FlowJo V10 software (BD Bioscience).
A list of all antibodies and dyes can be found in Supplementary Table
8
Quantifying Apoptosis and Cell Cycle
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DNA fragmentation and cell cycle were determined by using a hypotonic solution of propidium iodide (PI, [34 (link)]). After treatments, cells (1x105) were washed with PBS (pH 7.2) and stored in 95% ethanol overnight at −20 °C. Thereafter, cells were washed and incubated in a 400 μL solution containing PI (50 μg/ml), RNase A (100 μg/mL), EDTA (50 mM), triton X-100 (0.2%) for 30 min at 37 °C. The cell suspension was analyzed for PI fluorescence by using a BD LSRFortessa II flow cytometer (BD Biosciences). Quantitative data and figures were obtained using FlowJo 7.6.2 Data Analysis Software. Cells entering the sub-G1 phase were used as a marker of apoptosis (DNA fragmentation). For cell cycle analysis, the sub-G1 population was subtracted from the total acquired events, and the Dean Jett Fox analysis was applied (root mean square (RMS) < 10). The experiment was conducted three times, and 10,000 events were acquired for analysis.
9
Caspase-3 Positive Cells Quantification
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Flow cytometry acquisition was used to determine the percentage of Caspase-3 positive cells according to Ref. [37 (link)]. Jurkat cells were treated according to the above-mentioned procedures. The fixated Jurkat cells were washed and incubated with rabbit anti-Caspase-3 (Millipore, cat #AB3623) primary antibody (1:200) at 4 °C overnight. Cell suspensions were washed and incubated with DyLight 488 donkey anti-rabbit antibody (1:500). Finally, cells were washed and re-suspended in PBS for analysis on a BD LSRFortessa II flow cytometer (BD Biosciences). Twenty thousand events were acquired, and the acquisition analysis was performed using FlowJo 7.6.2 Data Analysis Software.
10
Multiparametric Flow Cytometry Analysis
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Trypsin was used to separate the cells after each treatment, and the cells were centrifuged for ten minutes at 2000 rpm. The cells were then permeabilized for 30 min using 1.5% BSA and 0.2% Triton X-100 after being cleaned with PBS. Next, primary antibodies were used against the N-terminal end of the bA4 polypeptide, phospho-TAU, oxidized DJ-1, p-c-Jun, and cleaved caspase 3 (Table 3 ) at 1:200. Secondary fluorescent antibodies were diluted 1:500 and incubated with the cells after a thorough rinse. Utilizing a BD LSRFortessa II flow cytometer (BD Biosciences), fluorescence analysis was carried out. Cells without primary antibodies served as a negative control. Ten thousand events were collected for evaluation, and FlowJo 7.6.2 data analysis software (TIBCO® Data Science) was used to produce quantitative data and visualizations. The cell population (found using forward scatter analysis, Y-axis) that exceeded the negative control’s baseline fluorescence (488 nm or 594 nm, X-axis) was used to perform the event analysis. As a result, event analysis was used to construct density plots, where cells in the quadrant indicated the cell population that fluoresced above the baseline.
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