Flow cytometry was used to investigate the cytotoxicity of Fe3O4 MNPs in Jurkat cells. Briefly, Jurkat cells (2.0×106 cells/mL) were cultured with the IC50 of Fe3O4 MNPs and incubated for 24, 48, and 72 hours. The cells were harvested by centrifugation at 1,500 rpm for 5 minutes and washed with phosphate-buffered saline. Next, 600 μL of 70% ice-cold ethanol were added to the cell pellets, which were then kept at −20°C overnight. A 1 mL volume of phosphate-buffered saline was added and spun down at 1,500 rpm for 5 minutes to remove the ethanol. The cell pellets were then washed twice with phosphate-buffered saline, stained with staining buffer containing 0.1% Triton X-100 (Sigma-Aldrich), 10 mM ethylenediaminetetraacetic acid (Sigma-Aldrich), 50 μg/mL Ribonuclease A (Sigma-Aldrich), and 3 μg/mL propidium iodide (Sigma-Aldrich), and incubated in the dark on ice for 30 minutes. Flow cytometry was performed with laser emitting excitation light at 488 nm using a FACSCalibur™ flow cytometer equipped with an argon laser (BD Biosciences). The data analysis was performed using CellQuest™ Pro software.
Argon laser
Manufactured by BD
Sourced in United States
The Argon laser is a type of continuous wave gas laser that generates light in the visible wavelength range. It operates by exciting argon gas using an electrical discharge, which produces a coherent beam of blue-green or blue-violet light. The core function of the Argon laser is to provide a stable and reliable source of high-intensity, monochromatic light for various scientific and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur flow cytometer, by BD (4 mentions)
Ribonuclease a, by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Facscalibur, by BD (1 mentions)
Staurosporine, by Thermo Fisher Scientific (1 mentions)
Facscalibur flow, by BD (1 mentions)
7 aad, by BD (1 mentions)
Annexin 5 pe, by BD (1 mentions)
Celltiter 96 aqueous one solution cell proliferation assay, by Promega (1 mentions)
Annexin 5 and 7 aad, by BD (1 mentions)
6 well non adherent plates, by Corning (1 mentions)
Cytofix, by BD (1 mentions)
Deadend fluorometric tunel system, by Promega (1 mentions)
Cellquest software, by BD (1 mentions)
Facsaria, by BD (1 mentions)
Facsdiva version 6, by BD (1 mentions)
11 protocols using argon laser
1
Cytotoxicity Evaluation of Fe3O4 MNPs
2
Annexin V Apoptosis Assay
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The apoptosis index was determined by annexin V labeling. Briefly, cells were harvested, washed and resuspended in the binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2 and 1.8 mM CaCl2) containing annexin V-PE and 7-AAD (BD Biosciences, USA). Apoptosis was analyzed on a FACScalibur flow cytometer equipped with an argon laser (BD Biosciences, USA) and quantified as the number of annexin V-PE positive and 7-AAD negative cells divided by the total number of cells. A minimum of 10,000 events was analyzed for each sample.
To confirm the inhibitory effects of activin A, in a second set of experiments, apoptosis was induced with 0.03 μM staurosporine (Invitrogen, USA) during the last 4 h of activin A treatment, following annexin V labeling.
To confirm the inhibitory effects of activin A, in a second set of experiments, apoptosis was induced with 0.03 μM staurosporine (Invitrogen, USA) during the last 4 h of activin A treatment, following annexin V labeling.
3
Assessing Cell Viability and Apoptosis
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Viability of cells cultured in 96-well plates at a density of 3,000 cells/well was measured after 24 h with a CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, USA) according to manufacturer’s protocol. In independent experiments, cell death was induced with 125 µM hydrogen peroxide for 1 h, followed by assessment of cell viability.
The cells were labeled with annexin V and 7-AAD (BD Biosciences, USA), and analyzed on a flow cytometer equipped with an argon laser (BD Biosciences, USA) for a minimum of 10,000 events for each sample for apoptosis assessment. Apoptotic cells were quantified as the number of annexin V-PE positive and 7-AAD negative cells divided by the total number of cells.
The cells were labeled with annexin V and 7-AAD (BD Biosciences, USA), and analyzed on a flow cytometer equipped with an argon laser (BD Biosciences, USA) for a minimum of 10,000 events for each sample for apoptosis assessment. Apoptotic cells were quantified as the number of annexin V-PE positive and 7-AAD negative cells divided by the total number of cells.
4
Tumor Cell Anoikis Assay in Non-Adherent Conditions
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To evaluate the tumor cell anoikis in non-adherent conditions, tumor cells (5 × 105) were cultured in non-adherent 6-well plates (Corning) for 8 hours. Tumor cells cultured in adherent conditions were used as a control. At the end of incubation, cells were carefully retrieved and analyzed by TUNEL staining for anoikis [12 (link)]. In brief, tumor cells were fixed with cytofix buffer (BD cytofix, BD Biosciences, San Jose, CA) for 15 minutes at 4°C, and then stored overnight in 70% ethanol at -20°C. The percentage of apoptotic cells were then evaluated using the APO-BRDU terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay according to the manufacturer’s instructions (Sigma, St. Louis, MO). Apoptotic tumor cells were quantitated by flow cytometry using an argon laser excited at 488 nm (BD Biosciences, San Jose, CA).
5
Annexin V-FITC and 7-AAD Apoptosis Assay
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The apoptosis index was determined using annexin V-FITC and 7-AAD labeling (BD Biosciences, United States) as previously described(Rodrigues et al., 2022 (link)). A minimum of 10,000 events were analyzed for each sample using a FACSCalibur flow cytometer equipped with an argon laser (BD Biosciences).
6
Apoptosis Evaluation in Jurkat Cells
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Apoptosis in Jurkat cells treated with the ZER-NLC was evaluated using the Tdt-mediated dUTP nick-end labeling (TUNEL) assay according to the manufacturer’s protocol (DeadEnd™ fluorometric TUNEL system; Promega, Madison, WI, USA) and analyzed in a FACSCalibur flow cytometer (BD) equipped with an argon laser (BD).
7
Annexin V-FITC Apoptosis Assay
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Apoptosis index was determined by annexin V-FITC labeling. The cells were collected, washed with PBS, and resuspended in the binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 1.8 mM CaCl2) containing annexin V-FITC at 1:500. After 20 minutes incubation in the dark at room temperature, the cells were also stained with propidium iodide (PI, Sigma-Aldrich). Apoptosis was analyzed on a FACSCalibur flow cytometer equipped with an argon laser (Becton Dickinson) and quantified as the number of annexin V-FITC positive and PI negative cells divided by the total number of cells. At least 10,000 events were analyzed in each sample.
8
Cell Cycle Analysis of SCC9 Cells
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For cell cycle analysis, SCC9 cells were seeded in 100-mm dishes and serum starved for 48 hours. Next, the cell culture medium supplemented with 10% FBS was added and the cells were collected after 24 hours. Cells were fixed in cold 70% ethanol, stored at −20°C, washed in cold PBS, and treated with 10 mg/mL of RNase for 1 hour at 37°C. After staining with 50 μg/mL of propidium iodide for 2 hours at 4°C, cell distribution in the cell cycle was analyzed by flow cytometry.
Cells were analyzed on a FACSCalibur flow cytometer equipped with an argon laser (Becton-Dickinson, San Jose, CA). At least 10,000 events were analyzed in each sample. Quantitative flow cytometric analysis was performed using CellQuest software (Becton-Dickinson).
Cells were analyzed on a FACSCalibur flow cytometer equipped with an argon laser (Becton-Dickinson, San Jose, CA). At least 10,000 events were analyzed in each sample. Quantitative flow cytometric analysis was performed using CellQuest software (Becton-Dickinson).
9
Identification of Human Hematopoietic Cells
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Fluorochrome-conjugated anti-human monoclonal antibodies were used to identify human hematopoietic cells (CD45-PE (phycoerythrin), human myeloid cells (CD13-APC (allophycocyanin). Human gp91phox expression was determined by indirect staining with murine monoclonal antibody 7D5 followed by FITC-conjugated goat anti-mouse immunoglobulin G antibody. For flow cytometry, a FACSsort (Argon laser; Becton Dickinson, San Jose, CA) was used and the data analyzed using Flowjo version 9.7.6 software.
10
Cell Cycle Analysis of Granulosa Cells
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Cell cycle analysis was performed by flow cytometry after staining the granulosa cells with propidium iodide (PI). Ovaries of sacrificed animals were collected and processed for flow cytometry as described previously (60) . A total of 10,000 cells were acquired for each experiment on BD FACS Aria with argon laser (Becton Dickinson; San Diego, CA). Data were analyzed using FACS Diva Version 6.1.3 software (BD Biosciences).
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