Cells were seeded at 2 × 105 cells per well in 6-well plates and incubated overnight at 37 °C, followed by the treatment as mentioned. Following the treatment, the cells were washed with PBS, collected with trypsin in 1.5 mL Eppendorf tubes and centrifuged at 2000 rpm, 4 °C, for 5 min. The lysates were suspended and stained with an FITC Annexin V Apoptosis Kit (BioLegend, San Diego, CA, USA). Annexin V-FITC and propidium iodide (PI) diluted with Annexin V binding buffer were used to stain the samples at room temperature for 15–30 min. After staining, the samples were transferred from Eppendorf to flow tubes and then 400 μL Annexin V binding buffer was added. Cell viability was evaluated with a flow cytometer (BD FACSCalibur, Franklin Lakes, NJ, USA) available for FL1 (Annexin V) and FL2 (PI) bivariate analyses. Analysis by calculating the percentage of the cells in the respective quadrants was performed using CellQuest PRO software 4.0.1.
Annexin 5 fitc apoptosis kit
Manufactured by BioLegend
Sourced in United States
The Annexin V-FITC Apoptosis Kit is a laboratory equipment product designed to detect and quantify apoptosis, a programmed cell death process. The kit utilizes fluorescein isothiocyanate (FITC)-conjugated Annexin V, a protein that binds to phosphatidylserine, which is exposed on the surface of apoptotic cells. This allows for the identification and analysis of cells undergoing apoptosis through flow cytometry or fluorescence microscopy.
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Lab products found in correlation
Facscalibur, by BD (3 mentions)
Facsaria 3, by BD (2 mentions)
5 and 6 chloromethyl 2 7 dichlorofluorescein diacetate acetyl ester cm h2 dcfda, by Merck Group (2 mentions)
Facscalibur cell analyzer, by BD (2 mentions)
Pd l1 apc, by BioLegend (1 mentions)
Facs lsr 2, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Pharmingen apc brdu flow kit, by BD (1 mentions)
Accuri c6 flow cytometer, by BD (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Facsverse, by BD (1 mentions)
Camptothecin, by Merck Group (1 mentions)
Novocyte flow cytometer, by Agilent Technologies (1 mentions)
Z vad fmk, by R&D Systems (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
Softmax pro 7, by Molecular Devices (1 mentions)
Bafilomycin a1, by Merck Group (1 mentions)
Wst 1 reagent, by Roche (1 mentions)
Versamax microplate reader, by Molecular Devices (1 mentions)
18 protocols using annexin 5 fitc apoptosis kit
1
Annexin V Apoptosis Assay
2
PD-L1 and Apoptosis Assay in C1498 Cells
3
Apoptosis Assay in 12-well Plate
4
Quantifying Apoptosis by Flow Cytometry
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After tomentosin treatment, % apoptosis rate was determined by a ow cytometry using FITC Annexin V apoptosis kit (BioLegend, 640.922) . Cell suspensions (1x10 6 cells/ml) were prepared from control and dose group cells with Dulbecco's PBS. And, 5 µl of FITC Annexin V and 5 µl of 7-ADD dye were added to 100 µl of cell suspensions. After incubation at room temperature for 15 minutes in the dark, 400 µl of Annexin V binding buffer was added to cell suspensions and % apoptotic cells were determined in a ow cytometer (BD FACSAriaTM III).
5
Quantifying Apoptosis by Flow Cytometry
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After tomentosin treatment, % apoptosis rate was determined by a ow cytometry using FITC Annexin V apoptosis kit (BioLegend, 640.922) . Cell suspensions (1x10 6 cells/ml) were prepared from control and dose group cells with Dulbecco's PBS. And, 5 µl of FITC Annexin V and 5 µl of 7-ADD dye were added to 100 µl of cell suspensions. After incubation at room temperature for 15 minutes in the dark, 400 µl of Annexin V binding buffer was added to cell suspensions and % apoptotic cells were determined in a ow cytometer (BD FACSAriaTM III).
6
Cell Cycle and Apoptosis Analysis
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Cells were incubated with 50 mM BrdU (BD Bioscience, Franklin Lakes, NJ, USA) for 2 h, and cell cycle analysis was performed using BD Pharmingen APC-BrdU Flow Kits according to the manufacturer's protocol (BD Bioscience, Franklin Lakes, NJ, USA). The samples were analyzed by flow cytometry on a BD Accuri C6 Flow Cytometer (BD Bioscience). Cell apoptosis was detected using the Annexin V-FITC Apoptosis Kit (Biolegend, San Diego, CA) according to the manufacturer's protocol. The cells were cultured for 48 h with normal medium and then treated with or without drugs for 24 h. Cells were collected and re-suspended in 1× binding buffer at a concentration of 1 × 106 cells per mL. Then, the cells (1 × 105) were incubated with 5 μL of Annexin V-FITC and 5 μL of PI for 15 min at 24 °C in the dark, followed by the addition of another 400 μL of 1× binding buffer. Samples were analyzed by flow cytometry. The data were analyzed using FlowJo software (San Francisco, CA, USA).
7
Evaluating Cellular Stress Response
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Major chemicals: The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Himedia. Dulbecco's Modified Eagle Medium (DMEM) high glucose media was purchased from Gibco, and 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate acetyl ester (CM-H2 DCFDA) was procured from Sigma. Annexin V-FITC apoptosis kit was used from Biolegend. The apoptosis-specific antibodies such as PARP and NRF2 (cell signaling technology), Bax, Bad, Bid, and Bcl2 were purchased from Santacruz Biotechnology. HDAC isoform antibodies were purchased from Cell Signaling Technology, and HRP-conjugated secondary mouse and rabbit antibodies were purchased from Santacruz Biotechnology.
8
Cell Cycle and Apoptosis Analysis of MKN28 Cells
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For cell cycle analysis using flow cytometry (FACSVerseTM, BD Biosciences, Franklin Lakes, NJ), MKN28 cells were plated at a density of 1 × 103 cells in 60mm culture dishes and then treated with BI-2536 (200 and 500 nM) for 72 h. Cells were then harvested and fixed in ice-cold 70% ethanol overnight at −20°C. Afterwards, cells were centrifuged at 300× g for 5 min, re-suspended in PBS containing 10 µg/mL of propidium iodide (PI) (P4170, Sigma-Aldrich, St. Louis, MO, USA), 100 µg/mL RNase A and 0.1% (v/v) Triton X-100 and incubated at 37 °C for 10 min. For cell apoptosis assay of MKN28 cells treated with BI-2536 (200 and 500 nM), we used the Annexin V-FITC Apoptosis Kit (#640914, BioLegend, San Diego, CA, USA) according to the manufacturer’s protocol. Briefly, a total of 1 × 103 cells was seeded in 60mm culture dishes for 24 h and then treated with BI-2536 for 72 h. Adherent and floating cells were then harvested, stained with Annexin V and PI for 15 min at room temperature and subjected to flow cytometry. The data were analyzed with FlowJo software.
9
Evaluating Cell Viability and Apoptosis
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Cells were treated with bafilomycin A1 (10 nM; Sigma‐Aldrich), z‐VAD‐FMK (20 μM; R&D Systems, Minneapolis, MN) and/or Torin‐1 (5 or 10 μM; Tocris/Bio‐Techne Ltd, Abingdon, OX, UK). All treatments were performed for 24 hr, except bafilomycin A1 for 6 hr. As control experiments, we included cells treated with DMSO or camptothecin (100 μM; Sigma‐Aldrich) and a combination of camptothecin (100 μM) and z‐VAD‐FMK (20 μM). For cell viability assay, 10 μl of WST‐1 reagent (Roche Diagnostics, Indianapolis, IN) was added into each well and incubated for 2 hr. Measurements were recorded at wavelengths 450 nm (WST‐1 cleavage products) and 690 nm (reference) using VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA) and analyzed with SoftMax Pro 7 (Molecular Devices). Five replicate wells were included for each treatment group. Relative cell viability in each treatment was normalized to DMSO‐treated group. All experiments were repeated three times independently. Cell apoptosis was evaluated using Annexin V‐FITC apoptosis kit (#640905; BioLegend, San Diego, CA). All staining conditions were performed according to the manufacturer's instructions and analyzed by NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA). All experiments were repeated four times independently.
10
Cell Viability and Apoptosis Assay
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The animal cell culture media (Dulbecco's Modified Eagle Medium with high glucose, DMEM) was purchased from Gibco. The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Himedia, and 5-(and-6)-chloromethyl-2′,7′-dichlorofluorescein diacetate acetyl ester (CM-H2 DCFDA) was procured from Sigma-Aldrich. Annexin V-FITC apoptosis kit was obtained from Bio legend. The apoptosis-specific antibodies against PARP (Cat no-9542T), Bax (Cat no-sc-70405), Bak (Cat no-556382,BD Bioscience), Bid (Cat no-sc-56025), Bcl2 (sc-7382), Caspase-9 (Cat no- sc-133109), HDAC-1(Cat no-34589S), HDAC-3(Cat no-85057S) isoform antibodies and actin (sc-47778) were purchased, antibodies HRP-conjugated secondary mouse (Cat no-sc-2357)and rabbit (Cat no-sc-2768) antibodies were purchased from Santacruz Biotechnology.
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