FACS analysis for cell cycle and apoptosis was done 72 hours post-transfection. The cells were harvested, washed with cold PBS, and resuspended in the nuclear stain DAPI for cell cycle analysis or stained with 7-AAD and Annexin-V-FITC using ANNEXIN V-FITC/7-AAD KIT (BD Biosciences) for apoptosis analysis according to the manufacturer’s protocol. Stained cells were immediately analyzed by FACS (BD FACSVerse; BD Biosciences). Cell viability was determined at 24, 48 and 72 h by using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI) according to the manufacturer’s protocol. Also a cytoselect 24-well cell migration and invasion assay kit (Cell Biolabs, Inc) was used for migration and invasion assays according to the manufacturer’s protocol. For migration and invasion assays two replicates were used and the experiment was repeated three times.
Annexin 5 fitc 7aad kit
Manufactured by BD
Sourced in United States
The Annexin V-FITC/7AAD kit is a laboratory tool used to detect and quantify apoptosis, or programmed cell death. It utilizes Annexin V, a protein that binds to phosphatidylserine, and 7-Aminoactinomycin D (7-AAD), a DNA-binding dye. This kit provides a method to identify and distinguish between viable, early apoptotic, and late apoptotic/necrotic cells through flow cytometry analysis.
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Lab products found in correlation
Celltiter 96 aqueous one solution cell proliferation assay kit, by Promega (2 mentions)
Facsverse, by BD (2 mentions)
Cytoselect 24 well cell migration and invasion assay kit, by Cell Biolabs (2 mentions)
Facscanto 2, by BD (2 mentions)
Propidium iodide rnase staining kit, by BD (2 mentions)
Facscanto, by BD (1 mentions)
Version 10, by FlowJo (1 mentions)
Lysis buffer, by Thermo Fisher Scientific (1 mentions)
Mouse cd4 t cell isolation kit, by Miltenyi Biotec (1 mentions)
Bcl2 associated x (bax), by BD (1 mentions)
Agarose, by Thermo Fisher Scientific (1 mentions)
Acridine orange base, by Merck Group (1 mentions)
3 4 5 dimethyl 2 thiazolyl 2 5 diphenyl 2h tetrazolium bromide, by Merck Group (1 mentions)
Sqstm1 p62, by Cell Signaling Technology (1 mentions)
Low melting agarose, by Sangon (1 mentions)
C myc, by Cell Signaling Technology (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
10 protocols using annexin 5 fitc 7aad kit
1
Cell Cycle, Apoptosis, and Migration Analysis
2
Apoptosis Quantification by Annexin V-FITC/7AAD
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The apoptosis rate was detected using the Annexin V-FITC/7AAD kit (BD Pharmingen, Franklin Lake, NJ, USA). Cells at the logarithmic growth stage were seeded at 1 × 105 cells/well with 2 mL medium in six-well plates and routine-cultured in an incubator at 37 °C with 95% humidity and 5% CO2 for 24 h. Cells were trypsinized and centrifuged at 300× g at 4 °C for 5 min. Then, the cells were washed with 1 mL precooled PBS and centrifuged. The above process was repeated twice. The cells were resuspended in 200 μL binding buffer and 5 μL 7AAD was added. After mixing gently, the cells were kept at room temperature for 15 min in the dark. Then, the cells were filtered using a 200-mesh filter screen and flow cytometry was performed within 1 h to detect the cell apoptosis rate.
3
Cell Cycle and Apoptosis Analysis by Flow Cytometry
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For cell cycle assay, cells were fixed by 75% ethanol for 24 h. After washing with phosphate buffer saline (PBS), cells were treated with RNase and stained with Propidium Iodide (PI). Finally, samples were detected on a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA). For apoptosis assay, cells were washed twice with cold PBS. Then, cell apoptosis were measured using the Annexin V-FITC/7-AAD Kit (BD Biosciences) according to the manufacturer's instructions. All data were analyzed using FlowJo7.6 software (TreeStar, San Carlos, CA, USA).
4
Quantification of Apoptotic Cells
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Quantification of apoptotic cells was conducted using flow cytometry. Briefly, A549 cells were pretreated with AB23A for 24 h and then were harvested and stained with an Annexin-V-FITC/7-AAD kit (BD Biosciences) according to the manufacturer's protocol. The data acquisition and analysis were performed with CellQuest 5.0 software (BD Biosciences).
5
Annexin V-FITC/7-AAD flow cytometry for apoptosis
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Cell apoptosis was detected using an Annexin V-FITC/7-AAD kit (BD Biosciences) according to the manufacturer's protocol, followed by flow cytometry (FACSCantoII; BD Biosciences). Flow data were analyzed using FlowJo version 10.0 (FlowJo LLC).
6
Cell Viability, Migration, and Apoptosis Assays
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Cell viability was determined at 24, 48 and 72 h by using the CellTiter 96 AQueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI, USA) according to the manufacturer's protocol. BioCoat control and Matrigel invasion chambers (BD Biosciences) and Cytoselect 24-well cell migration and invasion assay kits (Cell Biolabs, Inc., San Diego, CA, USA) were used for migration and invasion assays according to manufacturer's protocols. Fluorescence activated cell sorting (FACS) analysis was done 72 h post-transfection. The cells were harvested, washed with cold PBS, and resuspended in the nuclear stain DAPI for cell cycle analysis or stained with the 7-AAD and Annexin-V-FITC using ANNEXIN V-FITC/7-AAD KIT (BD Biosciences) for apoptosis analysis according to the manufacturer's protocol. Stained cells were immediately analyzed by FACS (BD FACSVerse; BD Biosciences).
7
Immunophenotyping and Apoptosis Analysis of T-Cells
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After red blood cells were lysed with lysis buffer (eBioscience, San Diego, CA, USA), the remaining cells were stained with fluorescein-labeled monoclonal antibody of CD4 (allophycocyanin), CD8 (peridinin-chlorophyll protein), CD25 (phycoerythrin), and CD69 (Fluorescein isothiocyanat). Splenocytes were harvested after mice were killed. The percentage of CD25−CD69+ and other subsets in CD4+ T-cells was determined by flow cytometry.
The CD4+ T-cells were isolated from splenocytes for Con A challenging in vitro. The isolation method was based on manufacturer’s protocol (mouse CD4+ T Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously.22 (link) After Con A (2.5 μg/mL) challenging and MGL (1 and 5 mg/mL) coculture for 0, 6, 12, and 24 hours, the CD4+ T-cells were harvested. The surface marker on CD4+ T-cells was analyzed by flow cytometry.
For detecting apoptosis in thymocytes, the thymus glands were collected and thymocytes suspension was prepared. The thymocytes were stained with CD4 and CD8 monoclonal antibody, and Annexin V-FITC/7AAD kit (BD Pharmingen, San Jose, CA, USA), before the flow cytometry analysis.
The CD4+ T-cells were isolated from splenocytes for Con A challenging in vitro. The isolation method was based on manufacturer’s protocol (mouse CD4+ T Cell Isolation Kit; Miltenyi Biotec, Bergisch Gladbach, Germany) as described previously.22 (link) After Con A (2.5 μg/mL) challenging and MGL (1 and 5 mg/mL) coculture for 0, 6, 12, and 24 hours, the CD4+ T-cells were harvested. The surface marker on CD4+ T-cells was analyzed by flow cytometry.
For detecting apoptosis in thymocytes, the thymus glands were collected and thymocytes suspension was prepared. The thymocytes were stained with CD4 and CD8 monoclonal antibody, and Annexin V-FITC/7AAD kit (BD Pharmingen, San Jose, CA, USA), before the flow cytometry analysis.
8
Evaluating Apoptosis Signaling Pathways
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AB23A (High pressure liquid chromatography ≥98%) was purchased from Shanghai Moqi Biological Technology Co., Ltd. (Shanghai, China). The Cell Counting Kit-8 (CCK-8; cat. no. C0039) was purchased from Beyotime Institute of Biotechnology (Haimen, China). The propidium iodide (PI)/RNase staining kit and the Annexin V-FITC/7AAD kit were all purchased from BD Biosciences (San Jose, CA, USA); All primary antibodies, including Bax (cat. no. ab53154; 1:1,000), Bcl-2 (cat. no. ab196495; 1:1,000), AKT (cat. no. ab38449; 1:1,000), phosphorylated (p)-AKT (cat. no. ab18206; 1:500), PI3K (cat. no. ab86714; 1:1,000), p-PI3K (cat. no. ab125633; 1:1,000), mTOR (cat. no. ab63552; 1:500), p-mTOR (cat. no. ab1093; 1:1,000) and GAPDH (cat. no. ab9484; 1:5,000), and horseradish peroxidase-conjugated anti-mouse IgG (cat. no. ab205719; 1:10,000) or anti-rabbit IgG (cat. no. ab205718; 1:5,000) secondary antibodies were purchased from Abcam (Cambridge, UK).
9
Evaluation of NL-101 Cytotoxicity and Mechanisms
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NL-101 was designed and synthesized by Hangzhou Minsheng Institute of Pharmaceutical Research. Dimethyl sulfoxide and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich (USA). Propidium iodide (PI)/RNase staining kit and Annexin V-FITC/7AAD kit were purchased from Becton Dickinson (San Diego, CA, USA). Acridine Orange base was purchased from Sigma-Aldrich (USA). Agarose was purchased from Invitrogen (USA). Low-melting Agarose was purchased from Sangon Biotech (Shang Hai, China). Antibodies against p21 (ab18209), γ-H2AX (ab11134), were purchased from Abcam (Cambridge, UK). Antibody against acetylated histone H3 was from Merck Millipore. Antibodies against histone H3 (db972), H2AX (db5729), and Chk2 (db928) were from Diagbio, and antibodies against caspase-3 (#9662), cleaved caspase-3 (Asp175, #9664), caspase-8 (#4790), caspase-9 (#9508), cleaved caspase-9 (Asp330, #9501), PARP (#9532), c-Myc (#9402), cyclin E1 (#4129), Cdk2 (#2546), ATG5 (#2630), SQSTM1/p62 (#5114), LC3B (#3868s), p-mTOR (#5536), mTOR (#2938), p-Akt (#4060), Akt (#4691), p-Chk2 (#12302), and β-actin (#4970) were purchased from Cell Signaling Technologies (Danvers, MA, USA). And horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad (Hercules, CA, USA).
10
Apoptosis and Necrosis Profiling
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Antibodies given in Additional file 13 : Table S7 were used for flow cytometry of surface antigens. The analysis was performed on a Becton–Dickinson FACS Canto II flow cytometer according to standard protocols. To specifically stain apoptotic and necrotic cells, the ANNEXIN V-FITC/7-AAD kit from Becton–Dickinson was used according to manufacturer’s instructions.
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