After co-culture of T lymphocyte CTLL-2 cells with neutrophils isolated from septic mice for 24 h, cells were collected for staining with APC-anti-CD3 antibody, FITC-Annexin V and PI (MultiSciences Biotech Co., Ltd.). T lymphocyte apoptosis was detected by flow cytometry as aforementioned and the apoptosis rate was calculated by FlowJo software, according to the manufacturer's instructions. Cells that stained positive for Annexin V were considered apoptotic.
Annexin 5 fitc
Manufactured by MultiSciences Biotech
Sourced in China
Annexin V-FITC is a fluorescently labeled protein that binds to phosphatidylserine, a component of the cell membrane. It is commonly used in flow cytometry and fluorescence microscopy applications to detect and quantify apoptosis or programmed cell death.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by MultiSciences Biotech (7 mentions)
Facscalibur, by BD (4 mentions)
Binding buffer, by MultiSciences Biotech (3 mentions)
Facsaria, by BD (2 mentions)
Facscanto 2, by BD (2 mentions)
Facscanto 2 flow cytometer, by BD (2 mentions)
Flow cytometry, by BD (2 mentions)
Dnase 1, by Merck Group (1 mentions)
Cd90 pe, by BioLegend (1 mentions)
Cd3 fitc, by BioLegend (1 mentions)
Cd8 pe, by BioLegend (1 mentions)
Cd11b fitc, by BioLegend (1 mentions)
Fitc lineage, by BioLegend (1 mentions)
Apc klrg1, by BioLegend (1 mentions)
Cd127 apc, by BioLegend (1 mentions)
Gr 1 apc, by BioLegend (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Monensin, by Thermo Fisher Scientific (1 mentions)
Percy5 cd45, by BioLegend (1 mentions)
Pe crth2, by BioLegend (1 mentions)
43 protocols using annexin 5 fitc
1
Septic Neutrophil-Induced T-Cell Apoptosis
2
Isolation and Analysis of Tumor-Infiltrating Cells
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Tumor tissues were minced and incubated in digestion solution containing 0.5 mg/ml collagenase V, 0.2 mg/ml hyaluronidase and 0.015 mg/ml DNase I (Sigma, St. Louis, USA) in a 37 °C water bath for 1 h.
The samples were then ltered through a 70 µm strainer before staining with uorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were rst stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were xed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.
The apoptosis kit FITC-Annexin V and APC-7-AAD (Multisciences, Hangzhou, China) was used following the manufacturer's protocols to analyze the apoptosis of CT26 cells.
The samples were then ltered through a 70 µm strainer before staining with uorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were rst stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were xed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.
The apoptosis kit FITC-Annexin V and APC-7-AAD (Multisciences, Hangzhou, China) was used following the manufacturer's protocols to analyze the apoptosis of CT26 cells.
3
Evaluating AVT-Induced Apoptosis in MM Cells
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MM cells were plated into a 96-well plate with a density of 5×10 4 cells per well, followed by the treatment of AVT for 24hrs. Cell growth and viability were assessed with the MTT assay as described previously 6 .To determine cell apoptosis, MM cells treated with AVT were stained with Annexin V-fluorescein isothiocyanate (Annexin V-FITC) and propidium iodide (PI) according to the manufacturer's instruction (MultiSciences Biotech Co., Ltd, Hangzhou, China). Stained cells were analyzed on a flow cytometer (FACSCalibur®, Becton Dickinson). Apoptosis was measured by flow cytometry as described previously 6 .
4
Apoptosis Assay in Macrophages
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Briefly, 5 × 106 macrophages were put into a 1.5 mL EP tube and mixed with 1× binding buffer (500 μL). Cells were then treated with 5 μL Annexin-V FITC and 10 μL PI (Multisciences, Hangzhou, China) and incubated for 5 min. The cells were tested using BD FACS ARIA (NJ, USA) and analyzed using Flow jo software.
5
Metformin Cytotoxicity on HeLa Cells
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HeLa cells were seeded in a 6-well plate (3 × 105 cells/well) in DMEM. HeLa cells were treated with metformin (0, 3, 12, and 24 mM) for 24 h. HeLa cells were fixed with precooled 4% paraformaldehyde for 15 min at room temperature, and 10 µL (5 µg/mL) of DAPI (Beyotime, Shanghai, China) solution was applied to each well and incubated for 1 h. Then the cells were immediately analyzed with a Cytation 1 Cell Imaging Multimode Reader (BioTek, VT, USA).
HeLa cells (3 × 105 cells/well) were seeded in 6-well plates in DMEM and treated with metformin (0, 3, 12, and 24 mM) for 24 h. The cells were collected after treatment with 0.25% trypsin (without EDTA), washed twice with PBS, and added to 500 µL of 1× binding buffer; the cell count was adjusted (1 × 104 cells/group), and the cells were stained with 5 µL of Annexin V-FITC and 10 µL of PI (MultiSciences, Hangzhou, China) in the dark at room temperature for 5 min prior to flow cytometry analysis (Millipore, Darmstadt, IN, USA). All experimental data were obtained from at least three independent measurements.
HeLa cells (3 × 105 cells/well) were seeded in 6-well plates in DMEM and treated with metformin (0, 3, 12, and 24 mM) for 24 h. The cells were collected after treatment with 0.25% trypsin (without EDTA), washed twice with PBS, and added to 500 µL of 1× binding buffer; the cell count was adjusted (1 × 104 cells/group), and the cells were stained with 5 µL of Annexin V-FITC and 10 µL of PI (MultiSciences, Hangzhou, China) in the dark at room temperature for 5 min prior to flow cytometry analysis (Millipore, Darmstadt, IN, USA). All experimental data were obtained from at least three independent measurements.
6
Cell Cycle and Apoptosis Analysis
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We analyzed the cell cycle of different treatment groups using the cell cycle testing kit (Multisciences, Hangzhou, China). Myoblasts were grown in six-well plates (1 × 106 cells/well) with 2 mL culture medium. After treatment for 24 hr, we harvested cells and washed them in PBS buffer. Then, 1 mL of DNA strain solution and 10 μL of permeabilization solution were added to the resuspended cells. After incubating for 30 min in the dark at room temperature, the cell suspension was used for flow cytometry (FACS Canto II, BD BioSciences, USA), and each treatment group had three independent replicates.
Cell apoptosis was measured by annexin V-FITC/PI staining assay. After incubation, cells with different treatment were washed three times with PBS buffer and then resuspended in 500 μL 1× binding buffer. Then, cells were incubated for 10 min in the dark at room temperature in the presence of annexin V-FITC (5 μL) and PI (10 μL; Multisciences, Hangzhou, China). Afterward, cells were analyzed using flow cytometry, and each treatment group had three independent replicates. Cells stained with only annexin V were evaluated as being in early apoptosis; cells stained with both annexin V and PI were evaluated as being in late apoptosis stage.
Cell apoptosis was measured by annexin V-FITC/PI staining assay. After incubation, cells with different treatment were washed three times with PBS buffer and then resuspended in 500 μL 1× binding buffer. Then, cells were incubated for 10 min in the dark at room temperature in the presence of annexin V-FITC (5 μL) and PI (10 μL; Multisciences, Hangzhou, China). Afterward, cells were analyzed using flow cytometry, and each treatment group had three independent replicates. Cells stained with only annexin V were evaluated as being in early apoptosis; cells stained with both annexin V and PI were evaluated as being in late apoptosis stage.
7
Evaluating Bacterial Adhesion on Superhydrophobic PDMS
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The experiments were divided into two groups, namely pristine PDMS control group and superhydrophobic PDMS (SHO-PDMS) group. E. coli and S. aureus were dissolved in 1 ml PBS respectively, then the dissolved bacteria were evenly added to the nutrient agar plate (NA), and cultured in a biochemical incubator at 37 °C for 24 h [33 ]. Disinfected PDMS membranes of appropriate size were placed in bacterial colony of NA for another 24 h. The PDMS were washed three times with PBS and stained with the Annexin V-FITC and the Propidium Iodide (PI) Staining Kit (MultiSciences, Hangzhou, China) according to the manufacturer's instructions. Bacterial density was observed and measured through a electron microscope.
8
GST-Induced Apoptosis in Cancer Cells
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BGC-823, MGC-803, SGC-7901 and GES-1 cells were seeded in six-well plates at a density of 1.2 × 10 6 cells per well, and each cell line was divided into a GST group and a control group with 3 parallel wells per group. Each well was treated with 20 µg/mL GST extract solution or sterile water. After 72 h, the cells in each group were collected and washed with PBS, and a single-cell suspension was prepared with the binding buffer provided with the kit. After being mixed with 5 µL of Annexin V-FITC (Multi Sciences, Hangzhou, China) and 10 µL of PI (Multi Sciences, Hangzhou, China), the cells were gently shaken for 5 min and then incubated for 30 min in the dark at room temperature. Flow cytometry performed on a FACSCalibur was used to detect cell apoptosis rates. Annexin V+/PIcells in the lower right quadrant (early apoptotic) and Annexin V+/PI+ cells in the upper right quadrant (late apoptotic) were counted to determine the number of apoptotic cells.
9
Apoptosis and Cell Cycle Analysis of LPS-Treated BENDs
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The effect of LPS (1.0 μg/ml) treatment BENDs on apoptosis and cell cycle was measured by flow cytometry. For cell apoptosis and necrosis, digested cells were stained by Annexin V-FITC and propidium iodide (Annexin V-PI) at 25°C for 15 min according to the manufacturer's instructions (Multisciences Biotech Co., Ltd., Hangzhou, China). For cell cycle assay, BENDs were harvested by trypsin digestion at the indicated time points (0 h, 6 h, 9 h, and 12 h) and fixed in ethanol at -20°C. Cells were then rehydrated and PI stained for 30 min at room temperature in the dark. Stained cells were examined using a flow cytometer (BD Biosciences, USA), and data were optimized using FlowJo software (Tree Star, USA).
10
Isolation and Characterization of IL-27Rα+ Cells
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Spleen cell suspension was prepared described as above, and cell number was adjusted into 1 × 106 cell/100 µL PBS. For cell staining, cells were treated with 1 µg anti‐Mouse CD3e PE‐Cyanine7‐ (eBioscience), 0.25 µg anti‐Mouse CD4 FITC‐ (eBioscience) and 1 µg anti‐IL‐27Rα/PE (Bioss)‐conjugated antibody in 30 minutes in dark, respectively. For apoptosis assay, cells were suspended with 0.5 mL binding buffer and incubated with 5 µL Annexin V‐FITC and 10 µL PI (MultiSciences) for 5 minutes in dark. The cells were washed for one time and samples were acquired on a Beckman counter CytoFLEX followed by data analysis using CytExpert software. For cell sorting, cells were treated with anti‐IL‐27Rα‐PE (R&D)‐conjugated antibody (0.25 µg/106 cells) for 30 minutes in dark. Wash cells for one time with sterility cold PBS buffer and sorted IL‐27Rα+ cells in Beckman MoFlo Astrios EQ counter.
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