After treatment with iso-suillin for 48 h, the cells were double stained with an Annexin V-FITC/PI Kit (MultiSciences Biotech, China) according to the manufacturer's instructions. The apoptosis rate and cell cycle distribution were analyzed using flow cytometry (Epics-XL II, Beckman Coulter, USA).
Epics xl 2
Manufactured by Beckman Coulter
Sourced in United States
The Epics-XL II is a flow cytometer designed for cell analysis and sorting. It is capable of detecting and measuring multiple parameters of individual cells or particles within a sample. The Epics-XL II provides researchers and clinicians with a tool for diverse applications in areas such as immunology, stem cell research, and cancer diagnostics.
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Lab products found in correlation
9 protocols using epics xl 2
1
Apoptosis and Cell Cycle Analysis
2
Flow Cytometric Analysis of Dendritic Cell Markers
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Formalin-fixed and fresh tissues were cut into sections, rubbed gently with plastic forceps and rinsed with physiological saline. The suspension was filtered with a 300-mesh copper grid to remove the block, and the cell suspension was centrifuged at 150 × g for 2 min. The cells (1×106/ml) were collected and washed twice in PBS. A flow cytometer (Epics-XL II; Beckman Coulter, Inc.) was used to determine the cell surface expression of CD1a, CD83, CD86 and CD80 according to previous protocol (13 (link),14 (link)). Briefly, mononuclear cells were stained using monoclonal antibodies to CD1a, CD83, CD86, CD80 (Beckman Coulter, Inc., Brea, CA, USA) and appropriate IgG isotype controls. Cells were kept at 4°C for 30 min, and filtered using a 500-mesh copper grid. The samples were analyzed using a FACSCalibur cytometer and CellQuest Pro software (version 5.1; BD Biosciences, Franklin Lakes, NJ, USA).
3
Podocyte Apoptosis Quantification
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The differentiated mouse podocytes were synchronized and stimulated with the indicated treatments for 12, 24, 48 and 72 h. The cells were collected and washed twice with 4°C normal saline. The supernatant was removed following centrifugation at 1,000 rpm for 5 min. The cells were then fixed with 70% ethanol overnight. The cells were centrifuged (1,000 rpm, 5 min), washed with normal saline, and then 1 ml DNA dye [propidium iodide (PI) 50 μg/ml, RNase 10 μg/ml and 1% Triton X-100; Sigma] was added. After staining for 30 min at 4°C, the cells were analyzed by flow cytometry (Epics-XL II; Beckman Coulter, Brea, CA, USA). Expo32ADC software was used for analysis: a hypo-diploid peak prior to the peak of the diploid cells was considered as the apoptotic cell population. The apoptotic rate was calculated according to the distribution histogram of the hypo-diploid population.
4
Quantification of TGF-β1 by Flow Cytometry
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To assess TGF-β1 amounts, 106 cells underwent incubation with PE-conjugated anti-mouse TGF-β1 antibodies (BioXCell, Lebanon, NH, USA) for 30 min at ambient, followed by filtration through a 500-mesh copper screen prior to flow cytometry analysis on an Epics-XLII (Beckman Coulter, Indianapolis, IN, USA). PE-rat IgG (BioXCell, Lebanon, NH, USA) was used as an isotype control.
5
Exogenous ROS Measurement in Renal Cells
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For exogenous ROS study, single cells of renal tissues were suspended in ice-cold PBS buffer. ROS was measured by staining the cells (Beyotime Biotechnology Company, Jiangsu, China) according to the manufacturer's protocol. After staining, cells were strained briefly and analyzed using Epics-XL II (Beckman-Coulter, USA). The intensity of ROS production by each individual cell was represented by mean fluorescence intensity (MFI).
6
Apoptosis Quantification by Flow Cytometry
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After transfection for 48 h, 1 × 106 cells in each group were cultured with 5% CO2 at 37°C for 48 h. The cells were collected after centrifugation at 2000 rpm for 5 min. Then the cells were collected again after mixing with 1 ml PBS without the supernatant, for two circles. With 1× binding buffer to adjust the cell density to 1 × 106 cells/ml, 100 μl cells were added to the flow tube, mixed with 10 μl Annexin-V-PE on ice, and reacted avoiding light for 15 min, then added with 380 μL of 1× binding buffer and 10 μl 7-ADD. The cells were detected using Epics-XL II flow cytometry (Beckman Coulter, Inc., Fullerton, CA, U.S.A.), and the number of apoptotic cells was calculated using CellQuest software by collecting 10000 cells. Apoptotic peak (Ap peak, G1 subpeak before G1) and cell circle were analyzed using ModFit software. The experiment was repeated three times.
7
Evaluating Iso-suillin Induced Apoptosis
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Cells were cultured, treated, and processed as described above. After culturing for 24 h, the collected cells were divided into different groups. For each experimental treatment, a total volume of 6 ml solution, which contained different concentrations and combinations of iso-suillin, Z-IETD-FMK (a caspase-8 inhibitor) (R&D, USA), and Z-LEHD-FMK (a caspase-9 inhibitor) (R&D) were added into the culture medium. The cells were cultured for another 48 h before double staining with the Annexin V-FITC/PI kit according to the manufacturer's instructions. The apoptosis rate was determined using flow cytometry (Epics-XL II, Beckman Coulter, USA).
8
Cell Cycle Analysis via Flow Cytometry
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Cells were centrifuged at 2,000 RPM/447.2 × g for 15 min at 37°C for collection and rinsed twice with PBS. Pre-chilled 70% ethanol was added and stored at 4°C overnight. Again, the cells were centrifuged at 2,000 RPM/447.2 × g for 15 min at 37°C and resuspended once in 1 ml PBS. Subsequently, 50 µg/ml propidium iodide, 100 µg/ml RNase A and 0.2% Triton X-100 PBS were added into 100 µl of a 1×106 cells/ml suspension. After a 30-min incubation at 4°C in the dark, the cell cycle was detected using a flow cytometer (Epics-XL II; Beckman Coulter, Inc., Brea, CA, USA) and analyzed using Expo 32 ADC XL4 (Beckman Coulter, Inc.). Experiments were repeated three times.
9
Annexin V Apoptosis Assay
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The apoptosis assay was performed using Annexin V Apoptosis Detection Kit (KeyGen, China). The single cell suspension of intestinal cells was prepared by enzyme digestion. The cells were washed twice using phosphate-buffered saline (PBS), re-suspended in Annexin V binding buffer, and the Annexin V-FITC and propidium iodide (PI) buffer were then added to the cells and incubated together in the dark at 4 °C for 10 min. Cell apoptosis was determined by flow cytometry (Epics-XLII, Beckman, USA).
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