Cells were stained for flow cytometry as described (14). Briefly, cells were stained for surface markers (30 min, 4°C, in the dark), fixed, permeabilized, stained for intracellular markers (30 min, 4°C, in the dark), washed, resuspended in a flow solution and analyzed (EPICS® XL-MCL cytometer with Expo32 software (Beckman Coulter). Anti-human mAb used: anti-FOXP3 conjugated to fluorescein isothiocyanate (FITC, clone PCH101) from eBiosciences); anti–CTLA-4–phycoerythrin (PE), anti-IL10-PE (clone 127107), and anti-TGFβ1-PE (clone 9016) from R&D Systems; and anti-CD3–phycoerythrin-cyanine 5 (PC5, clone UCHT1), anti-CD4− phycoerythrin-Texas Red conjugate (Energy Coupled Dye) (ECD, clone SFCI12T4D11), anti-CD4-PC5 (clone SFCI12T4D11), anti-CD25-PE (clone B1.49.9), and isotype controls from Beckman Coulter.
Epics xl mcl cytometer
Manufactured by Beckman Coulter
Sourced in United States
The EPICS XL-MCL cytometer is a flow cytometry instrument designed for cell analysis and sorting. It is capable of measuring and analyzing various cellular parameters, including size, granularity, and fluorescence. The EPICS XL-MCL is a compact and versatile instrument suitable for a wide range of applications in research and clinical settings.
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7 protocols using epics xl mcl cytometer
1
Multiparametric Flow Cytometry Analysis
2
Cell Cycle Analysis of Brentuximab Vedotin
3
Curcumin-Induced Cell Cycle Arrest
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Calu-1 cells were seeded in 100 mm dishes and the next day treated with 1 μmol/L or 5 μmol/L curcumin for 72 h. All attached and floating cells were washed with phosphate-buffered saline (PBS) and fixed in 0.125% formaldehyde followed by methanol. After incubation at −20°C, cells were washed with PBS and incubated for 45 min at 4°C in 50 μg/mL propidium iodide, 0.1% Nonidet P-40, 20 μg/mL RNase A, and 0.1% sodium azide in PBS. Propidium iodide fluorescence was measured on an EPICS XL-MCL cytometer (Beckman Coulter, Pasadena, CA).
4
Cell Cycle Analysis by Flow Cytometry
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The cells were centrifuged at 500 g for 10 minutes at 4ºC, washed twice with 1 ml icecold PBS and resuspended in 50 μl PBS. Following drop-wise addition of 1 ml ice-cold 75% ethanol, fixed cells were stored at -20ºC for at least 1 hour. Samples were then centrifuged and washed as above. The cell pellets were resuspended with 1 ml PBS containing 50 μg/ml propidium iodide and 100 μg/ml RNase A, and incubated for 1 h at room temperature. The cell cycle phase distribution was analyzed by flow cytometry using a Coulter EPICS XL-MCL cytometer (Beckman-Coulter, Fullerton, CA, USA) with an excitation setting of 488 nm. The propidium iodide-derived fluorescence was detected using a 620±15 nm band-pass filter. A minimum of 10,000 cells per experimental condition was evaluated. Cell debris was excluded from analysis. The cells with reduced DNA staining (hypodiploid cells), resulting from either fragmentation or decreased chromatin, were considered apoptotic cells.
5
Mitochondrial Membrane Potential Assay
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Cells were incubated with 10 µM of fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazolylcarbocyanine iodide (JC-1) for the last 30 min of recovery, collected by centrifugation at 500 g for 10 min at room temperature and washed with PBS. JC-1 is a cationic dye that accumulates and aggregates into energized mitochondria (high ∆m) in healthy cells.
After excitation JC-1 aggregates emit fluorescence at 590 nm. Upon cell injury, as mitochondrial membrane potential decreases, JC-1 aggregates are transformed back into JC-1 monomers, which emit fluorescence at 529 nm. Consequently, mitochondrial depolarization is indicated by a reduction in the red fluorescence. Cells were analyzed using a Coulter EPICS XL-MCL cytometer (Beckman-Coulter, Fullerton, CA, USA) with an excitation setting of 488 nm.
Monomers-and aggregates-derived fluorescence were detected using 525±20 nm and 620±15 nm band-pass filters. A minimum of 10,000 cells per experimental condition was evaluated. Cell debris was excluded from analysis. Cells treated with 10 µM of the depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 30 min were used as a positive control; in these conditions >60% of cells exhibited low mitochondrial membrane potential.
After excitation JC-1 aggregates emit fluorescence at 590 nm. Upon cell injury, as mitochondrial membrane potential decreases, JC-1 aggregates are transformed back into JC-1 monomers, which emit fluorescence at 529 nm. Consequently, mitochondrial depolarization is indicated by a reduction in the red fluorescence. Cells were analyzed using a Coulter EPICS XL-MCL cytometer (Beckman-Coulter, Fullerton, CA, USA) with an excitation setting of 488 nm.
Monomers-and aggregates-derived fluorescence were detected using 525±20 nm and 620±15 nm band-pass filters. A minimum of 10,000 cells per experimental condition was evaluated. Cell debris was excluded from analysis. Cells treated with 10 µM of the depolarizing agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) for 30 min were used as a positive control; in these conditions >60% of cells exhibited low mitochondrial membrane potential.
6
Phagocytic Activity Quantification
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Phagocytic activity of myeloid compartment was detected using the Phagotest kit (Opregen Pharma, Heidelberg, Germany).
Briefly, 100 μl heparinized whole peripheral blood is incubated with 20 μl opsonized FITC-labeled Escherichia coli for 10 min at 37°C in a water bath and in parallel a negative control sample remained on ice. The phagocytosis was stopped by placing the samples on ice. To eliminate the fluorescence of non-phagocytized bacteria, 100 μl of quenching solution were added. After a wash with 3 ml of washing solution (5 min, 250 × g, 4°C), the cells were incubated for 10 min on ice in 200 μl of DNA and analyzed by flow cytometry using a Coulter EPICS-XL-MCL cytometer.
Neutrophils were gated through the scatter parameters (forward, FCS vs side, SSC) and their green fluorescence histogram was analyzed. The phagocytic ability was expressed as percentage of fluorescent cells in the population studied and calculated by subtracting the percentage of the negative control sample (< 1%) from the positive sample.
Briefly, 100 μl heparinized whole peripheral blood is incubated with 20 μl opsonized FITC-labeled Escherichia coli for 10 min at 37°C in a water bath and in parallel a negative control sample remained on ice. The phagocytosis was stopped by placing the samples on ice. To eliminate the fluorescence of non-phagocytized bacteria, 100 μl of quenching solution were added. After a wash with 3 ml of washing solution (5 min, 250 × g, 4°C), the cells were incubated for 10 min on ice in 200 μl of DNA and analyzed by flow cytometry using a Coulter EPICS-XL-MCL cytometer.
Neutrophils were gated through the scatter parameters (forward, FCS vs side, SSC) and their green fluorescence histogram was analyzed. The phagocytic ability was expressed as percentage of fluorescent cells in the population studied and calculated by subtracting the percentage of the negative control sample (< 1%) from the positive sample.
7
Measuring Phagocytosis and Oxidative Burst
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Human peripheral blood leucocytes (3×106 cells/ml) were incubated in 200 μl RPMI 1640 medium containing 20% plasma, with either E. coli-FITC to measure phagocytosis or dihydrorhodamine 123 (DHR) to measure the production of H2O2, which oxidises DHR to fluorescent rhodamine 123. Cell suspensions were quenched with trypan blue 10%. Approximately 30,000 cells from each sample were analysed by flow cytometry, using a Coulter EPICS-XL-MCL cytometer (Coulter, Miami, FL, USA), selecting the polymorphonuclear cell population with the appropriate gating. Data was processed using the XL-2 software.
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