To measure apoptosis, SKOV3-TR cells (3 × 103) were seeded on black 96-well plates 24 hrs prior to the experiment. Liposomes were sterile filtered through 0.2-µm filters and incubated with the cells for 24 hrs and washed off. After a further 24 hrs, three fluorescent dyes: Hoechst 33342 (5 µg/mL), Yo-Pro (0.063 µg/mL) and propidium iodide (PI) (1 µg/mL) were added to stain live cell nuclei, cells in early apoptosis (slight membrane permeability), and late apoptosis/necrosis, respectively. After incubation at 37°C for 30 minutes, the cells were analyzed in situ using the iCyte® laser scanning cytometer (CompuCyte Corp. Westwood, MA)(39 (link), 40 (link)). Excitation/emission wavelengths used were 405/440 nm with a 30-nm bandwidth for Hoechst, 488/515 nm with a 30-nm bandwidth for Yo-Pro and 488/635 nm for propidium iodide. All data analyses were carried out using the iCyte software (Version 3.4). Alternatively, DNA content distributions were measured by propidium iodide staining. Upon treatment with liposomes (50 nM, PCT; 40 nM, XR) for 18 hrs, the cells were permeabilized by 70% ethanol overnight, stained with PI/RNase buffer (BD Biosciences), and detected by an LSR II Fortessa cell analyzer. The cell cycle distributions were further processed by FlowJo software (38 (link)).
Lsr 2 fortessa cell analyzer
Manufactured by BD
The BD LSR II Fortessa cell analyzer is a flow cytometry instrument designed for high-performance multiparameter analysis. It features advanced optics, sensitive detectors, and a flexible configuration to enable detailed cell characterization and sorting.
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Lab products found in correlation
Pi rnase buffer, by BD (1 mentions)
Anti cd86 fitc, by BD (1 mentions)
Fc receptor binding inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti cd163 pe, by BioLegend (1 mentions)
Anti cd80 apc, by BD (1 mentions)
Dcfh da, by Beyotime (1 mentions)
Bx471, by MedChemExpress (1 mentions)
Fc block, by Thermo Fisher Scientific (1 mentions)
Cd45 pe, by BD (1 mentions)
Cd8 fitc, by BD (1 mentions)
Cd4 apc, by BD (1 mentions)
Live dead stain, by Thermo Fisher Scientific (1 mentions)
Cd16 cd32, by Thermo Fisher Scientific (1 mentions)
Ficoll paque gradient, by Thermo Fisher Scientific (1 mentions)
Cellstripper solution, by Corning (1 mentions)
Click it edu alexa fluor 488 flow cytometry assay kit, by Thermo Fisher Scientific (1 mentions)
Saponin, by Merck Group (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Tetradecanoyl phorbol acetate, by Merck Group (1 mentions)
8 protocols using lsr 2 fortessa cell analyzer
1
Apoptosis Measurement in SKOV3-TR Cells
2
Antibody Staining for Flow Cytometry
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Antibody staining for flow cytometry was performed as described previously8 (link),11 (link),15 (link). Briefly, cells were subjected to a 1:200 dilution of primary antibodies for 20 min at 4 °C, washed with PBS +2% FCS (FACS buffer) and subjected to a secondary antibody staining if necessary. After two final washing steps, cells were resuspended in 100 μL FACS buffer and acquired on a LSR II Fortessa cell analyzer (BD Bioscience).
3
Flow Cytometric Analysis of Macrophage Surface Markers
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Macrophages were harvested, washed with PBS, then pelleted at 300 × g at 4°C for 5 min. Cells were blocked with 2% Fc Receptor Binding Inhibitor (eBioscience, Frankfurt, Germany) in PBS for 10 min on ice. Afterward, the following antibody mix was added in 100 ml PBS: anti-CD80-APC, anti-CD86-FITC (BD Biosciences, Heidelberg, Germany), anti-CD163-PE, and anti-CD206-PE-Cy5 (BioLegend, San Diego, CA, USA), followed by incubation for 20 min on ice in the dark. Samples were washed and analyzed by flow cytometry using an LSRII Fortessa cell analyzer (BD Biosciences).
4
Intracellular ROS Analysis in BM Cells
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Intracellular ROS analysis was performed using an oxidant-sensitive probe, DCFH-DA (Biyuntian, China). A total of 5 × 106 BM cells were loaded into flow tubes for the ROS detection. After staining with LSK antibodies, the cells were incubated with DCFH-DA in a water bath at 37 °C for 30 min. Fluorescence intensity was analyzed on a BD LSR II/Fortessa cell analyzer. Recombinant CCL-6 was purchased from Pepero Tech (Pepero Tech, USA). BX471 was purchased from MedChem Express (MCE, USA).
5
Murine Monocyte Isolation and Characterization
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Blood was collected by cardiac puncture with a 23G needle from male wild-type C57BL/6J mice into a 5 ml tube with 100 µl of 0.5 M EDTA and 3 ml DPBS. Separation was performed with a 1.077 g/ml Ficoll-Paque gradient (17-1440-02; Thermo Fisher Scientific). 2 ml of Ficoll gradient was layered with 4 ml of blood and spun at 300 ×g for 30 min at room temperature. The monocyte layer was transferred to a 15-ml falcon with 14 ml DPBS, spun at 300 ×g for 5 min at room temperature, repeated once. The pellet was then stained with LIVE/DEAD stain for 30 min (L34961; Thermo Fisher Scientific) and with antibodies CD16/CD32 (“Fc-block,” 14-0161-82; Thermo Fisher Scientific), CD45-PE (553081; BD), CD4-APC (553051; BD), and CD8-FITC (553030; BD), and analyzed with the LSR II Fortessa Cell Analyzer (BD).
6
Flow Cytometric Analysis of ABC Transporters
7
Quantifying Myoblast Proliferation via EdU Assay
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Proliferation rates in miR-501ΔMP myoblasts were measured using Click-iT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Invitrogen) according to manufacturer's instruction. Briefly, transfected cells were treated with 5 μM EdU for 6 h. Cells were trypsinized and washed with 1% BSA in PBS. Cells were then fixed, permeabilized, and stained with the click reaction using Alexa Fluor 488 azide. Flow cytometry was performed on the LSR II Fortessa cell analyzer (BD Biosciences). Analysis and determining the percentage of EdU-positive cells was performed using FlowJo software (v10.6.2, BD Biosciences).
8
Intracellular Cytokine Profiling by Flow Cytometry
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Intracellular cytokines were detected by flow cytometry. Briefly, T cells (1×106/mL) were stimulated with immobilized anti‐CD3 (1 μg/mL; OKT3, eBioscience) and tetradecanoylphorbol acetate (10 ng/mL; Sigma) for 6 hours in complete medium. Prior to the culture, the plates were centrifuged for 5 minutes at 800g. Three hours after activation, monensin (10 μg/mL; Sigma) was added. T cells were then collected, washed in phosphate‐buffered saline, and fixed with 2% formaldehyde. After fixation, T cells were permeabilized in phosphate‐buffered saline supplemented with 2% Fetal Calf serum and 0.5% saponin (Sigma). Permeabilized T cells were incubated with anti‐IL‐17, anti‐IL‐4, anti‐interferon‐γ, anti‐IL‐10, or anti‐FoxP3 monoclonal antibodies. All monoclonal antibodies were obtained from BD PharMingen. After washing, cells were analyzed using LSRII Fortessa™ cell analyzer (BD Biosciences, CA), and data were analyzed with Flowjo software. Quadrant markers were set accordingly to isotype‐matched controls.
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