Intracellular and mitochondrial ROS analyses were performed as previously described [37 (link)]. Briefly, the levels of intracellular ROS were determined by incubating cells for 10′ at 37 °C with the redox-sensitive probe 2′-7′-Dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Waltham, MA, USA) according to the instructions of the manufacturer. CM-H2DCFDA fluorescence was analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences) and data were processed with FlowJo software (Tree Star, Inc.). After treatments, cells were incubated with 5 µM MitoSOX Red (MitoSOX Red Mitochondrial Superoxide Indicator, Thermo Fisher Scientific Inc.) for 10 min at 37 °C and then 2.0 × 104 cells were acquired by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). Fluorescence data were processed with FlowJo software (Tree Star, Inc.).
Facs lsrfortessa x 20 cytofluorometer
Manufactured by BD
Sourced in United States
The BD LSRFortessaTM X-20 is a cytofluorometer manufactured by BD. It is designed to perform flow cytometry analysis. The device is capable of detecting and analyzing multiple parameters of cells or other particles suspended in a fluid stream.
Automatically generated - may contain errors
Lab products found in correlation
Mitosox red mitochondrial superoxide indicator, by Thermo Fisher Scientific (3 mentions)
3 hydroxy 1 2 dimethyl 4 1h pyridone deferiprone or l1, by Merck Group (2 mentions)
Cm h2dcfda, by Thermo Fisher Scientific (2 mentions)
Anti cxcr4 antibody, by Abcam (1 mentions)
Calcein acetoxymethyl ester ca am, by Merck Group (1 mentions)
Cm h2dcfda, by BD (1 mentions)
Mitosox red, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 annexin 5 dead cell apoptosis kit, by Thermo Fisher Scientific (1 mentions)
Ca am, by Merck Group (1 mentions)
Bodipy 581 591 c11 dye, by Thermo Fisher Scientific (1 mentions)
Calcein acetoxymethyl ester ca am calcein am, by Merck Group (1 mentions)
14 protocols using facs lsrfortessa x 20 cytofluorometer
1
Quantifying Intracellular and Mitochondrial ROS
2
CXCR4 Expression Analysis in K562 Cells
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K562 cells (2 × 105) were harvested and rinsed once. Then, the cells were incubated with anti-CXCR4 antibody (1:400; Abcam) for 1 h at 4°C. After primary antibody incubation, the cells were rinsed with 1X PBS and incubated for 30 min with Alexa Fluor 633 donkey anti-goat antibody (H+L) (1:400) resuspended in 5 mg/ml BSA and 0.76 mg/ml EDTA. The cells were rinsed with 1X PBS, resuspended in 300 μl 1X PBS and evaluated by a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences, San Jose, CA, USA).
3
Cellular Labile Iron Pool Measurement
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HEY and MCF7 cells were seeded in 6-well plates at a density of 5.0 × 105 cells/well and grown overnight, and treated at the concentration indicated for 24 h. Cells were loaded with 0.25 μM calcein acetoxymethyl ester (CA-AM) (Sigma-Aldrich, St. Louis, MO, USA) for 30 min at 37 °C, then washed with PBS1X and treated or not with 200 mM 3-hydroxy-1,2-dimethyl-4(1H)-pyridone (deferiprone or L1) (Sigma-Aldrich, St. Louis, MO, USA). Following staining, and washing with PBS, cells were analyzed using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The ∆ mean fluorescence intensity (∆MFI) between chelator-treated and untreated cells reflected the amount of LIP [54 (link)].
4
Annexin V-PI Assay for Apoptosis
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For identifying cells actively undergoing apoptosis, a double staining with Annexin V and PI was performed using Alexa Fluor®488 Annexin V/Dead Cell Apoptosis Kit (Thermo Fisher Scientific, Waltham, Massachusetts, United States) as previously described (Scicchitano et al., 2023 (link)). After staining, cells were incubated at room temperature for 15 min in the dark. Each tube was diluted with 400 μL of Annexin Binding Buffer and then cells were analyzed by flow cytometry using the FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Inc.). Three independent experiments were conducted.
5
Intracellular ROS Quantification by Flow Cytometry
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Intracellular ROS amounts were determined by incubating cells for 10 min at 37°C with the redox-sensitive probe 2′-7′-Dichlorodihydrofluorescein diacetate (CM-H2DCFDA; Thermo Fisher Scientific, Waltham, USA), according to the instructions of the manufacturer. CM-H2DCFDA fluorescence was analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences) and data were processed with FlowJo software (Tree Star, Inc.). Each experiment was performed in triplicate.
6
Quantifying Intracellular Labile Iron
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Intracellular labile iron concentration was determined by flow cytometry using the fluorescent iron sensor calcein acetoxymethyl ester (CA-AM). Briefly, cells were incubated with 0.25 μM CA-AM (Aldrich, Missouri, United States) for 30 min at 37°C in the dark. Then, cells were washed twice with PBS (1X) to remove the excess of CA-AM, and thus treated with 200 μM L1 (3-Hydroxy-1,2-dimethyl-4(1H)-pyridone, Sigma-Aldrich, Missouri, United States) or left untreated. The analysis was performed by FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). The difference in cellular fluorescence after and before incubation with L1 reflected the labile iron pool:
7
Flow Cytometric Analysis of PD-L1 Expression
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For the flow cytometry analysis of surface PD-L1, cells were incubated with PD-L1 antibody (anti-human CD274, APC, BioLegend, San Diego, California, United States) for 30 min in the dark. After washing twice with PBS (1X), cells were acquired in a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). Data were analyzed using FlowJo software (Tree Star, Inc.). Three independent experiments were conducted.
8
Mitochondrial Superoxide Detection by Flow
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Generation of mitochondrial ROS was measured by flow cytometry with the use of MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific Inc.). After treatments, cells were incubated with 5 µM MitoSOX Red for 10 min at 37°C, washed in PBS (1X), and then analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). A minimum of 20.000 cells was analyzed per condition. Fluorescence was measured using FlowJo software program (Tree Star, Inc.). Each experiment was performed in triplicate.
9
Mitochondrial Membrane Potential Analysis
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Changes in the mitochondrial membrane potential were analyzed by staining the cells with TMRE (tetramethylrhodamine ethyl ester) dye (Thermo Fisher Scientific, Waltham, USA). Briefly, cells were cultured in 6-well plates and, upon treatments, were incubated with 100 nM TMRE dye for 30 min at 37°C and then washed with PBS. Samples were then centrifuged at 1,000 r.p.m. for 3 min and the pellets were resuspended in 500 μl of PBS. TMRE fluorescence was analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). A minimum of 20,000 cells was analyzed per condition. Fluorescence was measured using FlowJo software program (Tree Star, Inc.). Each experiment was performed in triplicate.
10
Mitochondrial ROS Measurement by Flow
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Generation of mitochondrial ROS was measured by flow cytometry with the use of MitoSOX Red Mitochondrial Superoxide Indicator (Thermo Fisher Scientific Inc.). After treatments, cells were incubated with 5 µM MitoSOX Red for 10 min at 37°C and then analyzed by flow cytometry using a FACS BD LSRFortessaTM X-20 cytofluorometer (BD Biosciences). A minimum of 20,000 cells was analyzed per condition. Fluorescence was measured using FlowJo software program (Tree Star, Inc.). Each experiment was performed in triplicate.
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