Mitochondrial membrane potential (MMP) was analyzed using a JC-1 fluorescent probe kit (Beyotime Institute of Biotechnology, Haimen, China). The lipophilic and cationic fluorescent dye, JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazole carbocyanine iodide), is capable of selectively entering mitochondria, forming aggregates in mitochondria and emitting red fluorescence at high MMP. However, in low MMP, JC-1 cannot enter mitochondria and forms monomers that emit green fluorescence. The ratio of red to green fluorescence provides an estimate of change in MMP. The aggregates/monomer ratio was quantified at an excitation wavelength of 524 nm, emission wavelength of 590 nm (monomer, excitation at 490 nm and emission at 530 nm). HepG2 cells or L02 cells were co-cultured with SWNHs for 48 h in a 6-well plate and then treated with JC-1 (5 µg/ml) at 37°C for 30 min. Then, the cells were washed with JC-1 Staining Buffer (Beyotime Institute of Biotechnology) for three times. The fluorescence intensity was immediately measured using a fluorescence microscope at a magnification of ×200 (IX71; Olympus Corporation, Tokyo, Japan). The intensity of green and red fluorescence was measured using Image-Proplus 6.0 software (Media Cybernetics, Rockville, MD, USA).
Jc 1 fluorescent probe kit
Manufactured by Beyotime
Sourced in China
The JC-1 fluorescent probe kit is a laboratory tool used to measure mitochondrial membrane potential in cells. It contains the JC-1 dye, which exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green to red. This kit provides a method to assess changes in mitochondrial function.
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Lab products found in correlation
Jc 1 staining buffer, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Click it edu kit, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi, by Thermo Fisher Scientific (1 mentions)
Calcusyn software, by Biosoft (1 mentions)
Novocyte, by Agilent Technologies (1 mentions)
Reactive oxygen species assay kit, by Beyotime (1 mentions)
Axiovert 200, by Zeiss (1 mentions)
Rabbit monoclonal anti γh2a x antibody, by Cell Signaling Technology (1 mentions)
Fitc conjugated mouse anti rabbit igg antibody, by BD (1 mentions)
Annexin 5 apoptosis detection kit apc, by Thermo Fisher Scientific (1 mentions)
Annexin 5 fitc pi, by Thermo Fisher Scientific (1 mentions)
Novoexpress software, by Agilent Technologies (1 mentions)
5 protocols using jc 1 fluorescent probe kit
1
Mitochondrial Membrane Potential Analysis
2
Evaluating Apoptosis in DLBCL Cells
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To assess apoptosis, DLBCL cells were treated with different concentrations of ABT‐199 and Apatinib alone or in combination for 12 and 24 h, according to the manufacturer's instruction. DLBCL cells were harvested and then analysed by Novocyte (ACEA Bioscience, San Diego, CA, USA) after Annexin V/PI (Thermofisher, San Diego, CA, USA) staining for 15 min at room temperature in the dark. Primary DLBCL bone marrow or tissue samples were subjected to leukocyte separation. We used the Click‐iT EdU Kit (Thermofisher) to test the cell cycle. Loss of the mitochondrial membrane potential (MMP) (Δψm) was detected using JC‐1 Fluorescent Probe Kit (Beyotime Company, Shanghai, China). To evaluate the interaction between ABT‐199 and Apatinib, calcusyn software (Biosoft, Cambridge, UK) was used to calculate the combination index (CI). We used a Reactive Oxygen Species Assay Kit (Beyotime) as previously reported to detect the level of cellular reactive oxygen species (ROS), and the results were shown as the ratio of the mean fluorescence intensity.
3
Mitochondrial Membrane Potential Assay
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JC-1 fluorescent probe kit (Beyotime, Beijing, China) was used to determine Δψm. Briefly, cells were incubated in DMEM media containing JC-1 (5μg/ml) for 30min at 37°C, and were then measured by fluorescent microscope (Carl Zeiss, Axiovert 200). Δψm was determined by MPF-66 fluorescence spectrometer (Perkin-Elmer) with excitation wavelength at 490nm and emission wavelength at 530/590nm. The ratios of readings at according wavelengths were converted into relative Δψm values.
4
Apoptosis, Mitochondrial Potential, and ROS Assays in NALM-6/R Cells
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After treated for 48 h, 2 x 105 NALM-6/R cells were subjected to apoptosis assay using an Annexin V Apoptosis Detection Kit-APC (eBioscience Company, USA), following the manufacturer's instructions. For the mitochondrial membrane potential assessment, after different treatments for 48 h, 2 x 105 NALM-6/R cells were analyzed using a JC-1 fluorescent probe kit (Beyotime Company, China), following the manufacturer's instructions. To measure the intracellular reactive oxygen species (ROS) levels, about 2 x 105 NALM/R cells subjected to different treatments were washed in PBS buffer twice, and then incubated in 1 ml of serum-free RPMI 1640 medium containing 10 μM of H2DCFDA for 30 min at 37°C. The cells were harvested, and then washed in serum-free RPMI 1640 medium buffer twice to remove the remaining H2DCFDA. The fluorescent intensity was measured by flow cytometry. For assessing the degree of DNA damage, 2 x 105 NALM-6/R cells were incubated for 15 min on ice in hybridization buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.25% Triton X-100). After centrifugation, the cells were incubated with rabbit monoclonal anti-γH2A.X antibody (Cell Signaling Technology, USA) for 1 h, then washed with PBS and incubated with an FITC-conjugated mouse anti-rabbit IgG antibody (BD Pharmingen) for 30 min in the dark at room temperature.
5
Apoptosis and Mitochondrial Membrane Potential Assay
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To assess apoptosis, cells were treated with TPL and ABT-199 alone or in combination for 24 and 48 h, followed by staining with Annexin-V-FITC/PI (eBioscience, San Diego, California, USA) for 15 min at room temperature in the dark according to the manufacturer’s instructions. Cells were then analyzed by NovoCyte flow cytometry with NovoExpress software (ACEA Biosciences, Inc., USA) to determine the percentage of Annexin-V positive (apoptotic) cells. Loss of the mitochondrial membrane potential (Δψm) was measured using JC-1 Fluorescent Probe Kit (Beyotime Company, Shanghai, China) by following the manufacturer’s instructions as described previously15 (link). To assess the interaction between TPL and ABT-199, two separate approaches (i.e., CompuSyn and CalcuSyn) were used to calculate the combination index (CI). CI < 1.0 indicates synergism, while CI = 1.0 and CI > 1.0 indicate an additive or antagonistic effect, respectively.
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