Apoptosis and autophagy were evaluated with Olympus IX71 inverted microscope using Multi-Parameter Apoptosis Assay Kit (Cayman chemical, Ann Arbor, MI, USA, cat no 600330), respectively, Autophagy/Cytotoxicity Dual Staining Kit (Cayman chemical, Ann Arbor, MI, USA, cat no. 600140) according to the manufacturer’s protocol. Cells were cultured at a seeding density of 1 × 104 cells in 8-well chamber slides and transfected with 50 nM of miR-29b-3p inhibitor and negative control inhibitor for 48 h. To evaluate the apoptotic effect, cells were stained with Annexin-V FITC used to identify different types of cell death (early and late apoptosis) and evaluated at 485/535 nm. PI (propidium iodide), which is a DNA-binding dye molecule, is used to differentiate late apoptosis from necrosis being evaluated at 535/617 nm. The autophagic vacuoles were stained with monodansylcadaverine (MDC) and visualized in UV. Cellular death was evaluated at 520/610 nm using PI (propidium iodide).
Multi parameter apoptosis assay kit
Manufactured by Cayman Chemical
Sourced in United States, Estonia
The Multi-Parameter Apoptosis Assay Kit is a laboratory tool that measures multiple indicators of apoptosis, a type of programmed cell death, in a single experiment. The kit provides a comprehensive assessment of apoptosis through the simultaneous detection of various cellular parameters, including caspase activation, mitochondrial membrane potential, and cell membrane integrity.
Automatically generated - may contain errors
Lab products found in correlation
Ix71 inverted microscope, by Cayman Chemical (2 mentions)
Autophagy cytotoxicity dual staining kit, by Cayman Chemical (1 mentions)
Hoechst solution, by Thermo Fisher Scientific (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
Fascalibur, by BD (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
Cell cycle phase determination kit, by Cayman Chemical (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Dithiothreitol (dtt), by AppliChem (1 mentions)
Evos fl fluorescence microscope, by Thermo Fisher Scientific (1 mentions)
13 protocols using multi parameter apoptosis assay kit
1
Evaluating Apoptosis and Autophagy
2
Apoptosis Induction in Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded into a 24-well plate at 100,000 cells/well density in complete growth medium and were incubated for 24 hrs. Afterwards, medium was replaced with serum-free medium containing 1 μM unconjugated doxorubicin, [D-Lys(6)]LHRH), their combination, or AEZS-108. Twenty-four hours later, cells were trypsinized, collected into microcentrifuge tubes and the rate of apoptosis was measured by the Multi-Parameter Apoptosis Assay Kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions. Experiments were performed in triplicate. Values were expressed relative to the control.
3
Apoptosis Assessment using Multiparameter Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptotic effects were evaluated using the multi-parameter apoptosis assay kit (Cayman cat no 600330, Tallinn, Estonia) on an Olympus IX71 inverted microscope (Tokyo, Japan). Cells were cultured in 96-well plates at a seeding density of 1.2 × 104 and incubated for 24 h. Twenty-four hours later, the cells were treated with 10% serum. To evaluate apoptosis, we then double-stained the cells using the Hoechst solution (Invitrogen, cat no H3570) for the nuclei with ex/em at 361⁄497 nm and tetramethyl rhodamine ethyl ester (TMRE) (Invitrogen, cat no T669) for the mitochondrial membrane potential with ex/em at 560/595 nm. We used the Hoechst solution as a stain for apoptotic cells after it bonded to chromatin and produced a fluorescent signal. Thus, by using Hoechst we obtained images from a large area of the flask, allowing us to determine the percentage of the apoptotic and non-apoptotic cells using our fluorescent inverted microscope.
4
Assessing Mitochondrial Membrane Potential
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial membrane potential (MMP, Δψm) was assessed using Multi-Parameter Apoptosis Assay Kit (Cayman, Ann Arbor, MI). BaF3/T315I cells were treated with 1 μM HS-543 for 12 h. The cells were incubated with tetramethylrhodamine ethyl ester (TMRE), and DAPI staining at dark room condition. The cells were then suspended on poly-l-lysine-coated slides, followed by Shandon Cytospin 3 for 3 min at 1000 rpm. The slides were covered with DAKO before viewing with a confocal laser scanning microscope.
5
Multiparameter Apoptosis Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell death was detected using the Multi-Parameter Apoptosis Assay Kit (Cayman Chemical). Briefly, cells were counted and seeded in 96-well or 6-well plates. Cells without treatment or after treatment with the indicated drugs overnight were then washed with Cell-Based Assay Binding Buffer, and then incubated with Annexin V-FITC/7-Amino-Actinomycin D (7-AAD) double staining solution at room temperature in the dark for 10 minutes before resuspending in binding buffer for analysis. Fluorescence intensities were analyzed using SpectraMax M5 multi-detection microplate reader (Molecular Devices, Sunnyvale, CA) or on a FASCalibur flow cytometer (BD Bioscience, San Jose, CA) under FL1 (Annexin V-FITC) and FL3 (7-AAD) detectors. Annexin V-positive cells are defined as apoptotic, and Annexin V-7-AAD-double positive cells are defined as necrotic for flow cytometric analysis.
6
Multiparameter Apoptosis Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptosis was assessed using the Multi-Parameter Apoptosis Assay Kit (Cayman cat no 600330, Estonia) in accordance with the manufacturer’s protocol. The cells were plated at a density of 8 × 104 cells in 96-well plates and incubated overnight at 37 ∘C. Following this, the previously mentioned treatments were applied 24 h later, and cells were incubated for an additional 48 h at 37 ∘C under 5% CO2. For evaluation of apoptosis, cells were double-stained with tetramethyl rhodamine ethyl ester (TMRE) dye was used to assess mitochondrial membrane activity potential, while Hoechst dye was utilized for nuclear staining.
7
Cell Cycle and Apoptosis Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apoptosis and cell cycle phase in cells were determined using the Multi Parameter Apoptosis Assay Kit (Cayman Chemical, 601280) and the Cell Cycle Phase Determination Kit (Cayman Chemical, 10009349) following the manufacturer’s manual instructions. Stained cells were acquired and analyzed as described above.
8
Apoptosis Induction Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To evaluate the apoptosis induction, we performed Annexin V-fluorescein isothiocyanate (FITC) and tetramethylrhodamine, ethyl ester (TMRE) staining and analyzed the transfection results through fluorescence microscopy. Exactly 5×103 cells per well were cultured in a 96-well plate and reverse transfected with siRNA-p53 and siRNA-TNF. As a control, we used a scramble siRNA. After 24 hours of transfection, 20 μL of TMRE/Hoechst dye staining solution was added to each well and incubated for 15 minutes in a CO2 incubator at 37°C. Then, the cells were centrifuged for 5 minutes at 400× g and the supernatant discarded. Two hundred microliters of Binding Buffer with Annexin-V was added to each well and incubated for 10 minutes at room temperature. Cells were centrifuged for 5 minutes at 400× g and the supernatant discarded. One hundred microliters of Binding Buffer was added to each well and the cells were examined under a fluorescence microscope. In this assay, we used reagents from the Multi-Parameter Apoptosis Assay Kit (Cayman Chemical Company, Ann Arbor, MI, USA).
9
Mitochondrial Activity Evaluation using miR-29b-3p
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mitochondrial activity was evaluated with Olympus IX71 inverted microscope (20X magnification) using Multi-Parameter Apoptosis Assay Kit (Cayman chemical, Ann Arbor, MI, USA; catalog no. 601280). At a seeding density of 1 × 104, cells were seeded in 8-well chamber slide and transfected with 50 nM of miR-29b-3p inhibitor and negative control inhibitor for 48 h. Post-transfection, cells were stained with TMRE (Tetramethylrhodamine ethyl ester) and visualized at 560/595 nm to determine mitochondrial membrane activity potential. Additionally, cells were also stained with Hoechst and visualized in UV.
10
Mitochondrial Membrane Potential Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To determine mitochondrial membrane potential, the TMRE dye (maximal fluorescence wavelength: 595 nm) in the Multi-Parameter Apoptosis Assay Kit (Cayman Chemical, MI, USA) was used. TMRE is a cell-permeant, cationic, red-orange fluorescent dye that is readily sequestered by the active mitochondria. The membrane potential was thus preserved.
Briefly, Annexin V and TMRE staining was performed as follows. Cultured cells (2 × 10 5 ) were treated as necessary in a CO 2 incubator at 37 °C, with each sample in duplicate or triplicate. Cells were <80% confluent at the time of staining. The culture medium (DMEM) from each 35 mm diameter glass-based dish, as well as the cell layer, were not disturbed. Subsequently, 250 µL of the staining solution was added to each dish. The cells were incubated at room temperature in the dark for 15 min. The staining solution was carefully removed, and 500 µL of PBS (pH 7.4) was added. To the PBS, 2.0 mL of DMEM and 2 µL PI solution were added per dish.
Briefly, Annexin V and TMRE staining was performed as follows. Cultured cells (2 × 10 5 ) were treated as necessary in a CO 2 incubator at 37 °C, with each sample in duplicate or triplicate. Cells were <80% confluent at the time of staining. The culture medium (DMEM) from each 35 mm diameter glass-based dish, as well as the cell layer, were not disturbed. Subsequently, 250 µL of the staining solution was added to each dish. The cells were incubated at room temperature in the dark for 15 min. The staining solution was carefully removed, and 500 µL of PBS (pH 7.4) was added. To the PBS, 2.0 mL of DMEM and 2 µL PI solution were added per dish.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!