The cytotoxicity of free or drug-loaded HA/SiLN was evaluated using the standard MTT assay. SGC7901/ADR cells were seeded at 1.0×104 cells/well into 96-well plates and cultured until they reached 70%–80% confluence. The primary growth medium was replaced by 200 µL of fresh serum-free Dulbecco’s Modified Eagle’s Medium, to which free HA-SiLN or HA-SiLN/Q, HA-SiLN/D, HA-SiLN/QD was added to achieve various concentrations. The weight/weight (w/w) ratios of QC and DOX were 0.1, 0.2, 1, 5, and 10. Plates were then returned to the incubator for up to 48 hours of incubation. After this, 20 µL of 5 mg/mL MTT solution in PBS was added to each well for an additional 4 hours of incubation. Subsequently, the medium was carefully removed and replaced by 150 µL of DMSO and measured at 570 nm using a microplate reader (EL800; BioTek Instruments Inc., Winooski, VT, USA). Untreated cells were used as a control with 100% viability. The synergy effects of QC and DOX were calculated using combination index (CI).46 (link) In addition, apoptosis assay was performed using the Annexin V Apoptosis Detection Kit (Beyotime, Shanghai, People’s Republic of China) according to the manufacturer’s instructions.
Annexin 5 apoptosis detection kit
Manufactured by Beyotime
Sourced in China
The Annexin V Apoptosis Detection Kit is a laboratory tool used to detect and quantify apoptosis, a form of programmed cell death. The kit utilizes Annexin V, a protein that binds to phosphatidylserine, a component exposed on the cell surface during apoptosis. The kit provides a means to identify and analyze apoptotic cells.
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Lab products found in correlation
Facscalibur flow cytometer, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
El800, by Agilent Technologies (1 mentions)
Facscalibur system, by BD (1 mentions)
Cytoflex flow cytometer, by Beckman Coulter (1 mentions)
Accuri c6, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Facscan, by BD (1 mentions)
Huvec cells, by American Type Culture Collection (1 mentions)
Nuclear protein extraction kit, by Solarbio (1 mentions)
Dna content quantitation assay, by Solarbio (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Beyotime (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Solarbio (1 mentions)
Annexin 5 fitc and pi, by Beyotime (1 mentions)
Annexin 5, by BD (1 mentions)
Annexin 5 fitc, by Beyotime (1 mentions)
Fluorescence activated cell sorting (facs), by Beckman Coulter (1 mentions)
21 protocols using annexin 5 apoptosis detection kit
1
Evaluating HA/SiLN Cytotoxicity and Synergistic Effects
2
Doxorubicin-Induced Apoptosis Assay
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Doxorubicin was added to the cell medium at a final concentration of 1 μM 48 h after
transfection. After 24-h incubation, cells were collected and assayed with an Annexin
V Apoptosis Detection Kit (Beyotime, China) on a BD FACSCalibur™ System (Becton
Dickson, USA) following the manufacturer's instructions. Early apoptotic cells were
defined as Annexin-V-positive, propidium iodide-negative cells. Each experiment was
performed three times.
transfection. After 24-h incubation, cells were collected and assayed with an Annexin
V Apoptosis Detection Kit (Beyotime, China) on a BD FACSCalibur™ System (Becton
Dickson, USA) following the manufacturer's instructions. Early apoptotic cells were
defined as Annexin-V-positive, propidium iodide-negative cells. Each experiment was
performed three times.
3
Macrophage Phenotype and Apoptosis Analysis
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Flow cytometry was applied for examining CD206+/F4/80+and iNOS+/F4/80+ cell proportion of macrophages. After complete euthanization of mice, macrophages were isolated from femurs and resuspended in ice-cold PBS. Cells seeded in the 6-well plate were digested into single cells by trypsin. Half of the collected cells were incubated with PE-conjugated CD206 and AF 780-conjugated iNOS antibodies (Thermo Fisher Science, United States) for 45 min, respectively. Cells were further incubated with BV421-conjugated F4/80 antibody (Thermo Fisher Science, United States) for 45 min at 4°C. iNOS+F4/80+ macrophage was recognized as M1 phenotype and CD206+F4/80+ macrophage was recognized as M2 phenotype.
For apoptosis detection, Annexin-V Apoptosis Detection Kit (Beyotime, China) was used to detect apoptosis of BMMs. Cells were harvested with 0.25% trypsin and washed with PBS. Then cells were resuspended with 100 μL Annexin-V and propidium iodide (PI) staining buffer diluted in PBS for 20 min on the ice from light. The cells were analyzed on CytoFLEX Flow Cytometer (Beckman, IN, United States), and data were analyzed in FlowJo (Ashland, OR, United States) followed by instructions.
For apoptosis detection, Annexin-V Apoptosis Detection Kit (Beyotime, China) was used to detect apoptosis of BMMs. Cells were harvested with 0.25% trypsin and washed with PBS. Then cells were resuspended with 100 μL Annexin-V and propidium iodide (PI) staining buffer diluted in PBS for 20 min on the ice from light. The cells were analyzed on CytoFLEX Flow Cytometer (Beckman, IN, United States), and data were analyzed in FlowJo (Ashland, OR, United States) followed by instructions.
4
Annexin V Apoptosis Detection
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Cells were collected by trypsinization and washed with PBS. To detect cell death, an Annexin V apoptosis detection kit (Beyotime) was used. Briefly, 1 × 105 cells were mixed with Annexin V FITC and PI, and incubated for 15 min at room temperature in the dark. Stained cells were analyzed with a FACSCalibur flow cytometer (BD Biosciences).
5
Quantifying Apoptosis via Annexin-V Assay
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An Annexin-V Apoptosis Detection Kit (C1062L, Beyotime, China) was used to quantify the apoptotic rate according to the manufacturer’s instructions. Briefly, the cells were incubated with Annexin V-FITC and propidium iodide in the dark for 30 min, washed twice, and analyzed via flow cytometry (Accuri C6, BD Biosciences). The data were analyzed using FlowJo (version 10) software. The experiment was performed in triplicate, and statistical analysis was performed using GraphPad Prism software.
6
Annexin V-FITC Apoptosis Assay
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To evaluate apoptosis, after treatment, the HLE cells were determined by using the Annexin V–apoptosis detection kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions. The cells were incubated with Annexin-V–FITC and propidium iodide (PI) for 10 min, after which images of the intracellular Annexin V-FITC were captured using a fluorescence microscope (Leica DMi8).
7
Apoptosis Induction in IEC-6 Cells
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A total of 2 × 105 IEC-6 cells were plated in six-well plates and treated with JAC4 (1 μM) or DMSO for 24 h prior to X-ray irradiation exposure at 12 Gy. Twenty-four hours after the treatment, cells were harvested in PBS and analyzed with an Annexin V Apoptosis Detection kit (Beyotime) with a flow cytometer (BD, Franklin Lakes, NJ, USA).
8
Annexin V Apoptosis Detection Assay
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Cell apoptosis was detected by an Annexin V Apoptosis Detection Kit (Beyotime Biotechnology). At 36 hours after transfection, cells were collected followed by transfection by group. There were three treatment groups: (i) supplemented with PI; (ii) supplemented with annexin V‐FITC; and (iii) no supplementation. Cells were centrifuged and transferred into eppendorf tubes. Each tube contained 106 cells. Next, cells were washed twice with PBS, and then 100 μL binding buffer and 10 μg annexin V (20 μg/mL) marked with FITC were added for 30 minutes at room temperature. After antidark reaction with 5 μL PI (50 μg/mL) for 5 minutes, 400 μL binding buffer was added, and FACScan (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was used for quantitative detection. The group without Annexin V‐FITC or PI was taken as negative control group. FACS Diva software (Becton, Dickinson and Company) was used for data analysis.
9
Annexin V Apoptosis Detection in HepG2 Cells
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An Annexin V Apoptosis Detection kit (Beyotime Institute of Biotechnology, Shanghai, China) was used to analyze the effects of fatsioside A on the apoptosis of the HepG2 cells. Briefly, 1,000,000 HepG2 cells with the indicated treatment were stained with fluorescein isothiocyanate (FITC)-Annexin V and propidium iodide (PI). The early (annexin V+/PI−) and late (annexin V+/PI+) apoptotic cells were sorted using a fluorescence-activated cell sorting machine (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA). All experiments were performed in triplicate.
10
Phycocyanin Extraction and Cell Analysis
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The crude extract of R-phycocyanin was prepared on a laboratory scale from Porphyra haitanensis (the contents of crude protein 15.78 μg/mL). HUVEC cells were purchased from ATCC. Drosophila melanogaster was from Engineering Research Centre of Fujian-Taiwan Special Marine Food Processing and Nutrition.
Senescence-associated β-galactosidase (SA β-gal) staining, Hoechst and PI Staining Kit, Annexin V Apoptosis Detection Kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Cells counting kit-8 (CCK-8), the Mitochondrial Membrane Potential Assay Kit with JC-1, DNA content quantitation assay, and Nuclear Protein Extraction Kit were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). ECL was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
Senescence-associated β-galactosidase (SA β-gal) staining, Hoechst and PI Staining Kit, Annexin V Apoptosis Detection Kit, 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Cells counting kit-8 (CCK-8), the Mitochondrial Membrane Potential Assay Kit with JC-1, DNA content quantitation assay, and Nuclear Protein Extraction Kit were obtained from Solarbio Science & Technology Co., Ltd. (Beijing, China). ECL was purchased from Thermo Fisher Scientific (Waltham, MA, USA). All antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA).
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