Annexin V apoptosis detection kit (Life technologies, USA) was used for analysis of apoptosis. After indicated treatment, A549 and H1299 cells were trypsinized, collected, and resuspended. Approximately 2×105 cells were harvested and washed twice with cold phosphate buffer saline, then resuspended in 500 µL binding buffer. A total of 10 µL Annexin V-FITC and 10 µL propidium iodide were added to the solution and mixed well. After 15 minutes incubation, the cells were analyzed using flow cytometric analysis (BD Biosciences, San Jose, CA, USA).
Flow cytometric analysis
Manufactured by BD
Sourced in United States
Flow cytometric analysis is a technique that utilizes a specialized instrument, known as a flow cytometer, to analyze the physical and chemical characteristics of cells or particles suspended in a fluid stream. The flow cytometer measures and analyzes multiple parameters of individual cells or particles as they pass through a laser beam, providing quantitative data on various cellular properties such as size, granularity, and the presence of specific molecules or markers.
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Lab products found in correlation
Annexin 5 apoptosis detection kit, by Thermo Fisher Scientific (2 mentions)
Dcfh da, by Thermo Fisher Scientific (2 mentions)
Cellquest, by BD (1 mentions)
Facscan, by BD (1 mentions)
Apoptosis detection kit, by BD (1 mentions)
Fv1000, by Olympus (1 mentions)
Cd14 pe, by BioLegend (1 mentions)
Cd25 apc, by BioLegend (1 mentions)
Cd105 pe, by BioLegend (1 mentions)
Il 17, by BioLegend (1 mentions)
Cd19 percp cy5, by BioLegend (1 mentions)
Il 4 pe cy7, by BioLegend (1 mentions)
C11 bodipy dye, by Thermo Fisher Scientific (1 mentions)
Fcs express 6 flow cytometry, by De Novo Software (1 mentions)
Accuri c6 plus software, by BD (1 mentions)
Saponin, by BD (1 mentions)
Tcs sp5 confocal laser scanning microscope, by Leica (1 mentions)
Whole blood column kit, by Miltenyi Biotec (1 mentions)
Cryostor cs10 cryopreservation medium, by STEMCELL (1 mentions)
Cellxvivo human b cell expansion kit, by R&D Systems (1 mentions)
33 protocols using flow cytometric analysis
1
Annexin V-FITC Apoptosis Assay in A549 and H1299 Cells
2
Cell Cycle Analysis and Apoptosis Assay
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After the treatment, the cells were harvested, washed with ice-cold PBS, fixed with 70% ethanol at 4 °C for 30 min, and washed with ice-cold PBS. The cells were resuspended with a PI solution (0.3 mL) containing Triton X-100 (0.1% v/v), RNase (100 mg/mL) and PI (80 mg/mL) in the dark, and were analyzed with the FACScan and CellQuest software (Becton Dickinson, Mountain View, CA, USA). The population of apoptosis that contains sub-G1 DNA content was obtained as a percentage of the total number of cells. Furthermore, the apoptosis was determined using the staining with FITC-Annexin V/PI by an apoptosis detection kit (BD Pharmingen). The experiment was performed according to the manufacturer’s protocol. The apoptosis was detected and quantified using a flow cytometric analysis (BD Biosciences).
3
Flow Cytometric Analysis of EphA2 and CAR
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Flow cytometric analysis (BD, Mountain View, CA) was used to detect the expression of EphA2 on tumor cells and to detect the expression of CAR on EphA2-positive T cells. EphA2 expression in cell lines (A549, PC9, H1650, K562) was tested using a EphA2-PE antibody (BioLegend, San Diego, CA). Cells collected from pleural effusions were stained with both EphA2-PE antibody and CD45-APC antibody (BD, San Jose, CA). Cells were collected and washed once with phosphate buffered saline(PBS) containing 2% FBS prior to the addition of antibodies, and were incubated for 20 minutes on ice in the dark, washed twice prior to analysis.
4
Apoptosis Analysis of Osteoblasts
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After treatment as described above, osteoblasts and bone marrow-derived MSCs from each sample were stained using the Annexin V–FITC/PI kit, as described previously (Ding et al., 2012 (link); Cai et al., 2015 (link)). Then, flow cytometry was accomplished by flow cytometric analysis (BD, USA). The sample was additionally visualized under a confocal microscope (OLYMPUS FV1000, Japan). The Annexin V+/PI- osteoblasts were considered early apoptotic cells (Henry, Hollville & Martin, 2013 (link)). The Annexin V+/PI+ osteoblasts were considered late apoptotic cells (Henry, Hollville & Martin, 2013 (link)).
5
Immunophenotyping of GMSCs and Splenocytes
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GMSCs were collected and suspended in cell staining buffer (0.5% BSA in PBS with 2 mM EDTA) followed by incubation with CD14 (PE), CD19 (PerCP-Cy5.5), CD29 (APC), CD34 (PE), CD44 (FITC), CD45 (PE), CD73 (PE), CD90 (FITC), and CD105 (PE) antibodies (Biolegend) in the dark at room temperature for 30 min. For intracellular staining, splenocytes from mice were first stained with CD3 (APC/Cy7), CD4 (FITC), and CD25 (APC) antibodies (Biolegend) and then fixed, permeabilized, and stained intracellularly for IL-4 (PE/Cy7) and IL-17 (PE). After staining, cells were washed twice with PBS and submitted to flow cytometric analysis (BD). Data were analyzed using the FlowJo 7.6 software.
6
Lipid ROS Assessment via C11-BODIPY
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Lipid ROS were assessed using C11-BODIPY dye (Thermo Scientific, USA) according to the manufacturer’s protocol. Briefly, cells treated as indicated were collected and incubated with DMEM containing C11-BODIPY at a final concentration of 5 µM for 20 min at 37 °C. Then, the cells were washed twice with PBS to remove residual C11-BODIPY. Then, the filtered cell suspension was subjected to flow cytometric analysis (BD Biosciences, USA).
7
Annexin V-Based Apoptosis Detection
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An annexin V apoptosis detection kit (Thermo Fisher Scientific, Inc.) was used for the analysis of apoptosis. Following transfection, the cells were trypsinized, harvested and resuspended. A total of 2×105 cells were harvested and washed twice with cold PBS, and subsequently resuspended in 500 µl binding buffer. A total of 10 µl annexin V-fluorescein isothiocyanate and 10 µl propidium iodide was added to the solution and mixed well. Following 15 min of incubation, the cells were analyzed by flow cytometric analysis (BD Biosciences, San Jose, CA, USA) with FCS Express 6 flow cytometry software (FCS Express 6.04.0015, De Novo Software, Los Angeles, CA, USA).
8
Evaluation of Mitochondrial Function
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The effect of C. chinense stem extract and NPs on mitochondrial function was determined using the JC-1 staining method. For mitochondrial function, cancer cells (2.5 × 105 cells/well) were seeded into 6-well cultured plate for 24 h and treated with C. chinense stem extract (100, 250, and 500 µg/mL) and NPs (250, 500, and 1000 µg/mL) for 24 h. MCF-7 cells were harvested and washed, and the pellet cancer cells were collected. The cells were added to 100 µL of medium containing 5 µL of JC-1 assay reagents for 30 min at 37 °C in the dark; finally, 400 µL of DMEM medium was added. The mitochondrial membrane potential was determined through flow cytometric analysis (BD Biosciences, CA, USA) using BD Accuri C6 Plus software.
9
Quantifying Phosphorylation in Leukemia Cells
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Drug-treated K562 cells were fixed with 1% paraformaldehyde (BD Biosciences) for 30 min and permeabilized with 3% saponin (BD Biosciences) for 1.5 h (both at room temperature). Subsequently, the cells were stained using phycoerythrin-conjugated p-Tyr (1:1,000; cat. no. 558008) or p-CRK like proto-oncogene, adaptor protein (p-Crkl; 1:1,000; cat. no. 560788) antibodies (both BD Biosciences). Following washing with PBS, cells were recovered in 3% saponin and submitted to flow cytometric analysis (BD Biosciences), and mean fluorescence intensity (MFI) was recorded by FlowJo software (version 10) to observe the levels of p-Tyr and p-Crkl proteins.
10
Apoptosis Determination by Flow Cytometry
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Apoptosis was determined by flow cytometric analysis (BD Biosciences, Franklin Lakes, NJ, USA) with Annexin V-PE and 7-AAD double staining. After the cells were harvested and washed twice with ice-cold PBS, the cells were stained and fluorescence was measured at an excitation wavelength of 480 nm through the FL-1 filter (530 nm) and FL-2 filter (585 nm).
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