For analysis of BMCs within the hearts, a ‘myocyte-depleted’ cardiac cell population was prepared, incubating minced myocardium in 0.1% collagenase IV (GIBCO BrL, Carlsbad, CA, USA) 30 min. at 37°C, lethal to most adult mouse CMs. Cells were then filtered through a 70 mm mesh. To exclude spurious effects of enzymatic digestion, BM cells with or without collagenase treatment were stained revealing no significantly changed staining of labelled cell antigens. Mononuclear cells were separated by density-gradient centrifugation using 1.077 g/ml Histopaque solution (Sigma Chemicals, St. Louis, MO, USA), purified, and resuspended in PBS containing 1% BSA. Cells were incubated for 40 min. in the dark at 4°C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE) and peridininchlorophyll-protein (PerCP) conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, VLA-4-PE, c-kit-PE and CXCR4-PE (all from BD Pharmingen). Matching isotype antibodies (BD Pharmingen, San Jose, CA, USA) served as controls. Cells were analysed by three-colour flow cytometry using a Coulter® Epics®XL-MCLTM flow cytometer (Beckman Coulter, Cincinnati, OH, USA). Each analysis included 50.000 events.
Coulter epics xl mcltm flow cytometer
Manufactured by Beckman Coulter
Sourced in United States
The Coulter® Epics®XL-MCLTM flow cytometer is a laboratory instrument designed for cell analysis and sorting. It utilizes laser technology to detect and analyze the physical and fluorescent characteristics of cells or other particles suspended in a fluid stream. The Epics®XL-MCLTM provides high-speed data acquisition and analysis capabilities for a wide range of applications in the field of cell biology and research.
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Lab products found in correlation
Cd45 percp, by BD (3 mentions)
Cxcr4 pe, by BD (2 mentions)
Cd34 fitc, by BD (2 mentions)
Collagenase 4, by Thermo Fisher Scientific (2 mentions)
C kit pe, by BD (2 mentions)
Matching isotype antibodies, by BD (2 mentions)
Histopaque solution, by Merck Group (1 mentions)
Expo32 adc analysis software, by Beckman Coulter (1 mentions)
Cd4 pe, by BD (1 mentions)
Cd34 pe, by BD (1 mentions)
Cd31 pe, by BD (1 mentions)
Cd11b pe, by BD (1 mentions)
Cd86 pe, by BD (1 mentions)
F4 80 pe, by BD (1 mentions)
Cd133 pe, by BD (1 mentions)
Cd20 pe, by BD (1 mentions)
Cd11b biotin, by BD (1 mentions)
Sca 1 pe, by BD (1 mentions)
Cd45r b220 biotin, by BD (1 mentions)
Cd3 biotin, by BD (1 mentions)
4 protocols using coulter epics xl mcltm flow cytometer
1
Isolation and Characterization of Cardiac BMCs
2
Annexin V and Propidium Iodide Staining
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Cells were treated with angiotensin II for 24 hr, followed by propofol for 4 hr, and on the day of the experiment, they were trypsinized, washed once with ice-cold PBS and stained with annexin V and propidium iodide using a specific kit (BD Biosciences, Franklin Lakes, NJ, USA). Flow cytometry analysis of 20 000 cells per sample was performed on a COULTER EPICS XL-MCLTM flow cytometer (Beckman Coulter, Fullerton, CA, USA) and quantified using EXPO32 ADC analysis software (Beckman Coulter).
3
Cardiac Cell Characterization in Ischemic Mice
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Ten- to 12-week-old CXCR4-EGFP BAC transgenic reporter mice with or without LAD ligation were either treated with saline or DMOG (80 mg/kg/day) for up to 7 days. BM mononuclear and myocyte-depleted cardiac cells were separated as previously described [4 (link)]. Cells were incubated for 40 min in the dark at 4 °C with the following fluoresceinisothiocyanate (FITC)-, phycoerythrin (PE)-, and peridininchlorophyll-protein (PerCP)-conjugated monoclonal antibodies: CD45-PerCP, CD11b-PErCP, CD11b-PE, CD4-PE, CD20-PE, CD31-PE, CD34-PE, Flk-PE, CD86-PE, CD206-PE, F4/80-PE, CD133-PE, c-kit-PE, Sca-1-PE, CD3-biotin, CD45R/B220-biotin, CD11b-biotin, TER-119-biotin, and Ly-6G-biotin (all from BD Pharmingen). Matching isotype antibodies (BD Pharmingen) served as controls. Cells were analyzed by three-color flow cytometer using a Coulter Epics XL-MCLTM flow cytometer (Beckman Coulter). Each analysis included 50,000 events.
4
Cardiac Cell Populations Analysis
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We previously hypothesized that EPC may not be responsible only for the formation of new vessels but may also recruit local cells [13 (link)]. Therefore, we performed flow cytometry in order to evaluate the effects of cell transplantation on proangiogenic cardiac cell populations.
Hearts of the mice were investigated by flow cytometry (FACS) as described previously [19 (link)]. Briefly, for cardiac FACS analyses, infarcted hearts of the mice were explanted at day 2 and retrogradely perfused with saline (0.9% NaCl) to wash out circulating blood cells. Thereafter, a “myocyte-depleted” cardiac cell suspension was prepared, incubating minced myocardium in 0.1% collagenase IV (Gibco, Co Dublin, Ireland) for 30 min at 37 °C, lethal to most adult mouse cardiomyocytes. Cells from peripheral blood and hearts were incubated for 40 min in the dark at 4 °C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE), and peridininchlorophyll-protein (PerCP) conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, and CXCR4-PE (all from BD Pharmingen). A matching isotype antibody served as control. Cells were analyzed by 3-color flow cytometry using a Coulter® Epics® XL-MCLTM flow cytometer (Beckman Coulter, Brea, USA). Each analysis included 50,000 events.
Hearts of the mice were investigated by flow cytometry (FACS) as described previously [19 (link)]. Briefly, for cardiac FACS analyses, infarcted hearts of the mice were explanted at day 2 and retrogradely perfused with saline (0.9% NaCl) to wash out circulating blood cells. Thereafter, a “myocyte-depleted” cardiac cell suspension was prepared, incubating minced myocardium in 0.1% collagenase IV (Gibco, Co Dublin, Ireland) for 30 min at 37 °C, lethal to most adult mouse cardiomyocytes. Cells from peripheral blood and hearts were incubated for 40 min in the dark at 4 °C with the following fluoresceinisothiocyanate (FITC), phycoerythrin (PE), and peridininchlorophyll-protein (PerCP) conjugated monoclonal antibodies: CD45-PerCP, CD34-FITC, and CXCR4-PE (all from BD Pharmingen). A matching isotype antibody served as control. Cells were analyzed by 3-color flow cytometry using a Coulter® Epics® XL-MCLTM flow cytometer (Beckman Coulter, Brea, USA). Each analysis included 50,000 events.
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