Programmed cell death (PCD) detection was performed in single GFP-positive cells. Early (phosphatidylserine exposure) and late (membrane permeabilization) PCD stages were studied by Annexin V-PE Apoptosis Detection Kit I (BD Pharmingen) and flow cytometry (EPICs XL-MCL, Beckman Coulter). DNA fragmentation was studied in the end-stage PCD by TUNEL- (terminal deoxynucleotidyl transferase dUTP nick end labeling-) based APO-BRDU Kit (BD Pharmingen) and flow cytometry (LSRII, BD Biosciences). To avoid the overlap with GFP-marker, FITC-labeled anti-BRDU mAb was substituted with the PE-conjugated anti-BRDU mAb (BD Pharmingen). Results were analyzed as the mean ± SE, and compared using an independent two-sample t-test at P < 0.05 level of significance. Cell cycle analysis was performed in parental and transfected (vector-, WT/DN-ANXA7-, or p53-) LNCaP cells (18 h), using propidium iodide staining (Sigma-Aldrich) and flow cytometry (ModFit LT, Verity Software House and EPICs XL-MCL, Beckman Coulter).
Epics xl mcl
Manufactured by Beckman Coulter
Sourced in United States, Germany, Netherlands, France
The Epics XL-MCL is a flow cytometer system designed for advanced cellular analysis. It provides precise measurement and analysis of various cellular parameters, including size, granularity, and fluorescence intensity. The Epics XL-MCL is a versatile instrument suitable for a wide range of applications in the fields of immunology, cell biology, and hematology.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (7 mentions)
Expo32, by Beckman Coulter (6 mentions)
Expo32 adc software, by Beckman Coulter (3 mentions)
Sytox green, by Thermo Fisher Scientific (3 mentions)
Expo32 adc, by Beckman Coulter (3 mentions)
Hank s balanced salt solution, by Merck Group (3 mentions)
Fitc annexin 5 apoptosis detection kit, by BD (3 mentions)
H2dcfda, by Thermo Fisher Scientific (3 mentions)
Multicycle av software, by Phoenix Pharmaceuticals (2 mentions)
Rnase a, by Merck Group (2 mentions)
Fcs express version 3, by De Novo Software (2 mentions)
Cytomics fc500, by Beckman Coulter (2 mentions)
Annexin 5 fitc apoptosis detection kit, by Keygen Biotech (2 mentions)
Annexin 5 fluos staining kit, by Roche (2 mentions)
Anti cd4, by Beckman Coulter (2 mentions)
Gallios flow cytometer, by Beckman Coulter (2 mentions)
System 2 version 3.0 software, by Beckman Coulter (2 mentions)
Annexin 5 apoptosis kit, by BD (2 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (2 mentions)
Annexin 5 fluorescein isothiocyanate, by Beyotime (2 mentions)
190 protocols using epics xl mcl
1
Programmed Cell Death Analysis
2
Apoptosis and Cell Cycle Analysis of MWCNT-treated A549 Cells
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A549 cells were collected after MWCNT treatment, and washed twice with PBS. For apoptosis detection, the cells were suspended in 400 µl binding buffer and incubated with 5 µl Annexin V-FITC (MultiSciences, Hangzhou, China) and 5 µl PI in the dark at 37°C for 15 min. Cells were put on ice until analysis. The ratio of apoptotic cells was measured using a Beckman Coulter Epics XL-MCL device (Fullerton, CA, USA).
For cell cycle detection, cells were fixed in 70% ethanol for at least 2 h. After fixation, cells were centrifuged at 200 g for 5 min, and the cell pellet suspended in 500 µl propidium iodide (PI)/Triton X-100 staining solution containing RNase A for 15 min at 37°C. Cell cycle was assessed using a Beckman Coulter Epics XL-MCL device.
For cell cycle detection, cells were fixed in 70% ethanol for at least 2 h. After fixation, cells were centrifuged at 200 g for 5 min, and the cell pellet suspended in 500 µl propidium iodide (PI)/Triton X-100 staining solution containing RNase A for 15 min at 37°C. Cell cycle was assessed using a Beckman Coulter Epics XL-MCL device.
3
Comprehensive Cell Death Evaluation
4
Cell Cycle and Apoptosis Analysis
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The cell cycle distribution and the apoptosis rate were analyzed using flow cytometry as previously described.28 (link) After incubating with different concentrations of LR042 (0, 5, and 10 μM), LR042 (0, 5, and 10 μM), and 125I for 72 h, the cells were trypsinized, washed with PBS, and fixed with 70% ethanol overnight at 4 °C. The fixed cells were washed with PBS and stained with propidium iodide (PI) for 15 min in the dark. Then, the cell cycle arrest was analyzed using an Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, USA). The treated and untreated cells were trypsinized, washed with PBS, and costained with Annexin V and PI for 10 min, respectively. The apoptosis of cells was analyzed using an Epics XL-MCL flow cytometer (Beckman Coulter).
5
Annexin V Apoptosis Assay
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Cells were treated as indicated and incubated for 18 h at 37°C. Cells were collected, centrifuged at 200 × g for 5 min, supernatant discarded and the pellet was washed with 1× buffer. Cells were then resuspended in 90 μl of 1 × buffer and 10 μl of FITC-conjugated annexin V (Molecular Probes®, Grand Island, NY), incubated in the dark and analyzed using a Coulter® epics® XL-MCL.
6
Quantifying Neutrophil CD11b Expression
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Ten µL HDL of a tenfold concentrated stock solution was added to 90 µL PMNL suspension (0.3 × 106 cells/mL) and incubated for 30 min at 37 °C. Then, 10 µL PBS or N-formyl-methionyl-leucyl-phenylalanine (fMLP; Sigma-Aldrich Chemie GmbH, Steinheim, Germany) stock solution (10−7 M) was added and incubated for another 30 min at 37 °C. After addition of a fluorescence labelled monoclonal antibody (PC5-anti-CD11b; Immunotech Beckman Coulter, Marseille, France) the samples were incubated for 45 min at room temperature and put on ice. 500 µL ice cold PBS was added. Flow cytometry was performed on an Epics XL-MCL (Coulter, Hialeah, FL, USA). The CD11b surface expression was measured as mean fluorescence intensity (MFI).
7
Measuring CD14 Expression on PMNLs
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Ten microliters of HDL of a tenfold concentrated stock solution was added to 90 µL PMNL suspension (0.3 × 106 cells/mL). After incubation for 30 min at 37°, 10 µL PBS or fMLP (Sigma-Aldrich Chemie GmbH, Steinheim, Germany) stock solution (10−7 M) was added and incubated for a further 30 min at 37 °C. The samples were incubated for 45 min at room temperature in the presence of a fluorescence-labelled monoclonal antibody (ECD-anti-CD14; Immunotech Beckman Coulter, Marseille, France) and then placed on ice. After addition of 500 µL ice cold PBS, flow cytometry was performed on an Epics XL-MCL (Coulter, Hialeah, FL, USA). The surface expression was measured as percentage of CD14 positive cells.
8
DNA Content Analysis of Malignant Tissues
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Coulter DNA Prep Kit (Beckman Coulter) was used for quantitative measurements of DNA content in malignant tissues. Fluorescence-stained samples were analyzed by flow cytometry with a Coulter Epics XL-MCL flow cytometer and data were evaluated with MultiCycle AV Software (Phoenix Flow Systems). A minimum of 1×104 events were analyzed for each sample. DNA determinations provided information on the percentage of cells in the S phase of the cycle, which was subsequently used as a marker of cellular proliferation among malignant tissues.
9
Vitronectin-Mediated Complement Activation
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To analyze whether vitronectin bound to the microbial surface is functionally active, the microbial pathogens were incubated with vitronectin purified from human plasma (10–50 μg/ml) (Corning, Kaiserslautern, Germany) for 30 min at 37°C. After three washes, microbes were incubated with C5b-6 (1 μg/ml) and C7 (1 μg/ml) for 5 min, and thereafter C8 (0.4 μg/ml) and C9 (1 μg/ml) were added for 30 min at 37°C. All these complement factors were purchased from Complement Technology (Tyler, TE). After washing, deposited C5b-9 was detected by mouse anti-human C5b-9 mAb (Abcam, Cambridge, UK) followed by Alexa fluor-647-conjugated polyclonal goat anti-mouse pAb (Abcam). After two additional washes, microbes were analyzed by flow cytometry (EPICS XL-MCL; Coulter, Hialeah, FL). All incubations were kept in a final volume of 100 μl PBS-BSA and washings were done with the same buffer. Primary and secondary pAb were added separately as negative controls for each strain analyzed.
10
Characterization of Mesenchymal Stem Cells
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MSCs were characterized at the second passage. The cells
were incubated with antibodies against CD45 PC5, CD73
PE, CD105 FITC, and NG2 PE. A total of 20,000 cells/
sample at a flow rate of approximately 200 cell events/s were
recorded to obtain fluorescence histograms. A Coulter Epics
XL-MCL was used during the experiments and the data
were analyzed using EXPO 32 ADC software (Beckman
Coulter Inc., USA). Flow cytometry analysis revealed that
there were significant expressions of CD105, CD73, and
NG2, which are specific to MSC antigens, while there was
no detection of CD45, which is specific to the hematopoietic
marker antigen (Figure2 ). These results showed that these
cells are MSCs.
were incubated with antibodies against CD45 PC5, CD73
PE, CD105 FITC, and NG2 PE. A total of 20,000 cells/
sample at a flow rate of approximately 200 cell events/s were
recorded to obtain fluorescence histograms. A Coulter Epics
XL-MCL was used during the experiments and the data
were analyzed using EXPO 32 ADC software (Beckman
Coulter Inc., USA). Flow cytometry analysis revealed that
there were significant expressions of CD105, CD73, and
NG2, which are specific to MSC antigens, while there was
no detection of CD45, which is specific to the hematopoietic
marker antigen (Figure
cells are MSCs.
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