A mouse corneal keratocyte cell line was transfected with agomir-122 (20 μM), antagomir-122 (20 μM), or a plasmid carrying CPEB1 cDNA. Alternatively, cells were transfected with a plasmid carrying CPEB1 cDNA with wild-type or miR-122-binding site-mutated 3′-UTR. The cells were then treated with or without a mixed cytokine cocktail containing 100 ng/ml TNF-α, IL-1β and TNF-γ (Peprotech, Rocky Hill, NJ, USA). After 48 h, the cells were stained with annexin V (Keygen Biotech, Nanjing, China) per the manufacturer’s instructions, and the percentage of apoptotic cells (annexin V+) was determined by flow cytometry.
Annexin 5
Manufactured by Keygen Biotech
Sourced in China
Annexin V is a calcium-dependent phospholipid-binding protein that has a high affinity for phosphatidylserine (PS). It is commonly used as a tool in flow cytometry and fluorescence microscopy for the detection and quantification of apoptosis.
Automatically generated - may contain errors
Lab products found in correlation
Facscalibur, by BD (4 mentions)
Propidium iodide, by Keygen Biotech (4 mentions)
Facscalibur flow cytometer, by BD (3 mentions)
Propidium iodide, by Merck Group (2 mentions)
Sirtinol, by Merck Group (2 mentions)
Specific monoclonal anti sirt1 antibody, by Abcam (2 mentions)
Il 1β, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Tnf γ, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Rh123, by Dojindo Laboratories (1 mentions)
Sodium hydrosulfide, by Merck Group (1 mentions)
Caspase 3 activity detection kit, by Beyotime (1 mentions)
Lsr 2, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
Cellquest software, by BD (1 mentions)
Lsr flow cytometer, by BD (1 mentions)
Annexin binding buffer, by Thermo Fisher Scientific (1 mentions)
Fcap array software, by BD (1 mentions)
31 protocols using annexin 5
1
Regulation of Corneal Keratocyte Apoptosis
2
SIRT1 Inhibition and Cell Senescence
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Sodium hydrosulfide, homocystein, sirtinol, dimethyl sulfoxide (DMSO), and propidium iodide (PI) were purchased from Sigma (St. Louis, MO, USA). Rh123 was supplied by Dojindo Molecular Technologies, Inc. (Rockvile, MD, USA). Specific monoclonal anti-SIRT1 antibody was obtained from Abcam (Hong Kong, China). Specific monoclonal antibodies of P16INK4a, P21CIPL were purchased from OriGene Biotech Inc. (Burlingame, UK). Annexin V was bought from Nanjing Key GEN Biotech Co., Ltd. (Nanjing, China). DMEM medium, horse serum and fetal bovine serum were supplied by Gibico, BRL (Ground Island, NY, USA).
3
Apoptosis Analysis of NPMSCs
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Following compression for 48 h, the control and treated NPMSCs underwent trypsinization and centrifugation and were washed with ice-cold PBS and resuspended in 500 μl of 1x binding buffer. 5 μl propidium iodide (PI) and 5 μl Annexin-V (Nanjing Keygen Biotech, Nanjing, China) were then added, and cells were incubated in the dark at room temperature for 15 min. The Annexin and PI double-positive percentage (indicating late apoptosis cells) was measured using flow cytometry (BD LSRII, Becton Dickinson). In addition, NPMSCs were seeded in 6-well culture plates and treated as described above. For each group, cells were rinsed in PBS three times and incubated for 5 min in the dark at room temperature in 5 μmol/l Annexin-V and 5 μmol/l PI. The stained cells were then imaged under a laser scanning confocal microscope (LSM, Zeiss, Germany).
The activity of caspase-3 was measured using a Caspase-3 Activity Detection Kit (Beyotime, China). Briefly, after treatment, both the attached and the floated NPMSCs were totally collected. Next, the supernatant samples were used to evaluate the caspase-3 activity strictly according to the manufacturer's protocols. Finally, caspase-3 activity normalized and the total protein in each group was detected by evaluating the optical density at a wavelength of 405 nm.
The activity of caspase-3 was measured using a Caspase-3 Activity Detection Kit (Beyotime, China). Briefly, after treatment, both the attached and the floated NPMSCs were totally collected. Next, the supernatant samples were used to evaluate the caspase-3 activity strictly according to the manufacturer's protocols. Finally, caspase-3 activity normalized and the total protein in each group was detected by evaluating the optical density at a wavelength of 405 nm.
4
Annexin V Staining of Podocytes
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Podocytes from different groups were quantified by annexin V staining according to the manufacturer’s instructions (KeyGEN Biotech, Jiangsu, China). Podocytes were harvested and washed twice with phosphate-buffered saline (PBS). The cells were resuspended in 100 μl of ice-cold binding buffer and incubated with 5 μl of annexin V (conjugated with FITC) for 15 min in the dark. After resuspension in 400 μl of binding buffer, the cells were observed under a fluorescence microscope (Olympus, BX53, Tokyo, Japan).
5
Annexin V and PI Cytofluorometry
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6
Apoptosis Detection by Flow Cytometry
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Cells were digested using EDTA-free trypsin, washed twice with Phosphate Buffered Saline (PBS), resuspended by adding 500 µl of Binding Buffer, incubated together with 5 µl of Annexin V and 5 µl of PI (KeyGEN BioTECH), for 15 min at room temperature, and analyzed for apoptosis by flow cytometry.
7
Apoptosis and Cell Cycle Analysis
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Cells were cultured and exposed to the tested compounds for 24 h. For apoptosis analysis, cells were harvested and washed once with PBS and then resuspended and incubated in propidium iodide (PI)/Annexin V solution (KeyGen Biotech). At least 10,000 live cells were analyzed on a flow cytometer (BD Accuri™ C6; BD Biosciences, San Jose, CA, USA). For cell cycle analysis, cells were harvested and fixed in 75% cold ethanol at −20°C overnight. Cells were then resuspended and incubated in PI solution. DNA content was determined using a flow cytometer.
8
Annexin V-PI Apoptosis Assay in C2C12 Cells
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A total of 1 × 106 C2C12 cells of each group were collected and washed twice using the incubation buffer containing 10 mmol/L HEPES/NaOH (pH 7.4), 140 mmol/L NaCl, and 5 mmol/L CaCl2 [22 (link)]. Next, C2C12 cells were resuspended in 150 μL of phosphate-buffered solution (PBS) including 1.5 μg/mL Annexin V (KeyGen, Nanjing, Jiangsu, China) and propidium iodide (PI) (Thermo Fisher Scientific). Then, the cells were incubated with Annexin V and PI in dark at room temperature (RT) for 15 min. After washing with PBS, C2C12 cells were resuspended again in 400 μL incubation buffer and analyzed using flow cytometry (BD FACSCalibur, BD Biosciences, San Jose, CA, USA).
9
Quantifying Apoptosis with Annexin V
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Cells were digested with trypsin containing no EDTA and centrifuged for 5 min at 2000 r/min before washing twice with pre-cooled PBS. Cells were collected by centrifugation, and 1 × 10 6 cells were transferred into Eppendorf (EP) tubes. Into each EP tube, 500-µL binding buffer, 5-µL propidium iodide (PI), and 5-µL Annexin V (Jiangsu KeyGEN BioTECH Corp., China) were added. After adding staining material, the EP tube was incubated for 10-15 min away from light. Cell suspension was filtered with 400-mesh cell strainer to remove cell masses and placed on ice. Within 30 min, flow cytometry detection was conducted to analyze cell apoptosis. The experiment was repeated three times.
10
Detecting Apoptosis in Transfected Cells
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Apoptosis in transfected cells was analyzed by flow cytometry using a Becton Dickinson LSR flow cytometer with CellQuest software (BD Biosciences, San Diego, CA, USA). We used 300 nM H2O2 for 24 h to induce apoptosis after transfection for 48 h, and then cells were collected and resuspended in 500 μL of binding buffer of every tube. Next, 5 μL of annexin V and 5 μL of propidium iodide (Keygen Biotech, Nanjing, China) were added to every tube for 15 min in the dark simultaneously or separately (as the control to debug the machine) and then analyzed to identify the late apoptotic cells, early apoptotic cells, and viable cells, and the proportion of apoptotic cells was calculated.
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