Flow cytometry can detect early apoptosis and late apoptosis or necrosis of cells by Propidium iodide (PI) and Annexin V (Invitrogen, Carlsbad, CA) staining. Cells were seeded in six-well plates. When cells grew to about 80% confluence, they were treated with20, 40, 60μg/mL Epoxycytochalasin H for 24h. Then cells were collected and stained with propidium iodide (PI) and Annexin V to analyze apoptosis by guava easyCyte flow cytometry (Merck, USA).
Guava easycyte flow cytometry
Manufactured by Merck Group
Sourced in United States, Germany
The Guava® easyCyte flow cytometry system is a compact, benchtop instrument designed for analyte detection and analysis. It utilizes flow cytometry technology to rapidly measure and characterize multiple parameters of individual cells or particles in a sample.
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Lab products found in correlation
Annexin 5, by Thermo Fisher Scientific (1 mentions)
Annexin 5, by Merck Group (1 mentions)
Mitochondrial membrane potential assay kit with jc 1, by Beyotime (1 mentions)
Apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Thermo Fisher Scientific (1 mentions)
Fluorescence microscope, by Zeiss (1 mentions)
Guavasoft 2, by Merck Group (1 mentions)
Gibco cell dissociation buffer, by Thermo Fisher Scientific (1 mentions)
Flow express software, by De Novo Software (1 mentions)
13 protocols using guava easycyte flow cytometry
1
Apoptosis Detection by Flow Cytometry
2
Epoxycytochalasin H Mitochondrial Potential
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Mitochondrial membrane potential were measured using Mitochondrial membrane potential assay kit with JC-1 (Beyotime Biotech,China). Cells were seeded in six-well plates. When cells grew to about 80% confluence, they were treated with20, 40, 60μg/mL Epoxycytochalasin H for 24h. Then cells were collected and stained with JC-1 to analyze mitochondrial membrane potential by guava easyCyte flow cytometry (Merck, USA).
3
Apoptosis Assay with Fisetin Nanocarriers
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The apoptosis assay was carried out using the Annexin V FITC-Apoptosis Detection Kit (eBioscience™, San Diego, California, USA). Briefly, EA.hy926 and 3LL cell lines were seeded onto 24-well plates at densities of 500,000 cells/well and 200,000 cells/well, respectively. After incubation for 24 h at 37 °C, under 5% CO2, the cells were exposed to 50 μM of Fisetin NCs, 50 μM of free Fisetin, DMSO, and P407 for 24 h, respectively. Untreated cells were grown as negative control groups. The supernatant was removed and then harvested with 250 μL/well of Trypsin-EDTA (0.05%), followed by centrifugation at 2000 rpm for 5 min. Annexin V-FITC and Propidium Iodide (were equally mixed v:v into the binding buffer (Apoptosis Detection Kit, eBioscience™, San Diego, California, USA) and kept in the dark for further use. All the samples were incubated with 110 μL of the above mixture for 15 min and the reactions were stopped by adding 300 μL of binding buffer. The cells were then analyzed by using Guava EasyCyte™ flow cytometry (Merck Millipore, bioscience, Guyancourt, France).
4
Flow Cytometry Analysis of LDLR Expression
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Flow cytometry analysis of the LDLR expressed on the cell surface was conducted as previously described [41] (link), with slight modification. Following incubation with opti-MEM for 12 h, HepG2 cells were treated with 20 μg/ml hPCSK9 alone or co-treated with 20 μg/ml anti-PCSK9 mAbs for 12 h. Then, the cells were digested with trypsin, detached by scraping and washed with PBS and collected in a 1.5 ml tube, then fixed in 200 μl of 4% (w/v) paraformaldehyde in PBS for 5 min at room temperature. Cells were incubated with 200 μl of 0.1% Tween in PBS (PBS-T) and blocked with 10% goat serum in 0.3 M glycine in PBS for 30 min, then incubated with rabbit anti-LDLR monoclonal antibody (Cat# ab52818, 1:100) for 30 min at room temperature, and followed by incubation with Alexa Fluor® 488-conjugated goat anti-rabbit IgG (Cat# D110061, 1:200) for 30 min at room temperature. After washing, the detection was performed directly on a Guava EasyCyte™ Flow Cytometry (Merck Millipore, Germany) at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. The levels of LDLR on the cell surface were analyzed by using FlowJo software 7.6 (FlowJo, Oregon, USA) with 10,000 cells.
5
Quantification of GFP Expression
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In vitro expression of the GFP was measured and photographed at the magnification of ×20 using a fluorescence microscope (Zeiss, Germany), 24 and 48 h post-transfection. Quantification of the green fluorescence was assessed by flow cytometry. The cells were washed in PBS, dissociated with 200 µl of 0.25% trypsin and re-suspended in a total volume of 1 ml following the addition of 800 µl PBS containing 10% FBS. The cells were used immediately subjected to the Guava easyCyte™ Flow Cytometry (Merck Millipore, Germany).
6
Evaluating Apoptosis and Necrosis with Annexin V-FITC
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Apoptotic and necrotic were evaluated using Annexin V-FITC apoptosis Kit (Thermo Fisher Sciencetific, Waltham, MA, USA). Cells were treated with ovalitenone at concentrations 100 and 200 µM, and incubated at 37 °C for 24, 48 and 72 h. Cells were suspended with 100 µL of 1 × binding buffer and incubated in 5 and 1 µL of PI in the dark at room temperature for 15 min. Binging buffer added 400 µL of incubation buffer, and cells were analyzed using guava easyCyteTM flow cytometry (Merk, DA, Germany).
7
Norcycloartocarpin Induces Apoptosis in Lung Cancer Cells
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A549, H460, H23, and H292 cells were seeded onto 96-well plates and incubated at 37°C for overnight. Cells were treated with norcycloartocarpin at concentrations 10, 25, and 50 μM, and incubated at 37°C for 24 h. Apoptotic and necrotic were determined using Annexin V-FITC/PI apoptosis kit (Thermo Fisher Sciencetific, Waltham, MA, USA). Cells were suspended with 100 μl of 1× binding buffer and incubated with 5 μl of PI in the dark at room temperature for 15 min. Subsequently, binging buffer added 400 μl of incubation buffer, and cells were analyzed using guava easyCyteTM flow cytometry (Merk, DA, Germany).
8
Apoptosis induction by germacrone
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Eca109 and EC9706 cells were seeded in 6-well plates at a density of 1 × 105 cells/well. After 24 h cultivation, cells were treated with different concentrations (0, 10, 20, 30 μg/mL) of germacrone for 24 h. Cells were then preprocessed and submitted to Annexin V/propidium iodide (PI) staining for FACS analysis according to the protocol described previously [23 (link)]. Briefly, cells were harvested by centrifugation (2000 rpm for 5–10 min) and resuspended in 300 μL of 1 × Binding Buffer. Subsequently, 5 μL of Annexin V-FITC and 5 μL of PI solution were added followed by incubation at room temperature for 15 min in the dark. Induction of apoptosis was measured by a Guava easyCyte flow cytometry (Millipore, MA, USA) and analyzed using FlowJo software.
9
Cell Death Assay Using Flow Cytometry
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For cell death assay, MDCK cells were stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s protocol (HaiGene, Harbin, China) and then evaluated for apoptosis using flow cytometry. Briefly, MDCK cells seeded in 60 mm dish (including both adherent and floating cells) were trypsinized with trypsin and resuspended in 2 mL MEM. The harvested cells were centrifuged at 1500 rpm for 5 min, and washed twice with PBS. Cell pellets were resuspended in 1 × Annexin V binding buffer at a final concentration of 2 × 105 cells/mL and then stained with 5 μL of Annexin V-FITC and 8 μL of PI in 1 × Annexin V binding buffer for 10 min at room temperature in the dark. The apoptotic cells were determined using Guava® easyCyte flow cytometry with GuavaSoft 2.5 (Millipore, Darmstadt, Germany). This experiment was performed in triplicate. Percentage of total cell death (% cell death) = (number of both apoptotic and necrotic cells/number of all cells) × 100%.
10
Quantifying Hydrogen Peroxide Using DCFH-DA
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To measure the levels of reactive oxygen species (ROS), the fluorescent probe 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) was utilized, which is a commonly employed method for quantifying hydrogen peroxide (H2O2). The oxidation-insensitive DCF was used as a control to ensure that changes in uptake, ester cleavage, and efflux of the probe had not occurred. There was no significant alteration in the fluorescence of cells labeled with the oxidation-insensitive probe. A2780 cells were plated in 60 mm culture dishes with 2 mL of RPMI medium at a density of 1.0 × 105 cells/mL per dish. Following a 24 h incubation period, the cells were harvested through centrifugation after being treated with SMM extracts at specific time intervals. Subsequently, the cells were resuspended in PBS and stained with 20 μM DCFH-DA. The fluorescence intensity was then assessed using Guava® easyCyte flow cytometry (Millipore).
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