For lymphocytes differentiation, blood samples were harvested at indicated time points and monocytes were separated though isodensity centrifugation. Thereafter, approximately 106 cells were re suspended in 100 μL of PBS and incubated with 2 μg anti-CD3c FITC together with anti-CD4 PE or anti-CD8 PE antibodies (all from BioLegend, San Diego, CA, USA) for 20 min on ice. Thereafter, the cells were washed twice with PBS and flow cytometry was performed using a FACSort instrument (BD Biosciences, San Jose, CA, USA). Data was analyzed using FlowJo software (Tree Star, San Caros, CA, USA).
Facsort instrument
Manufactured by BD
Sourced in United States
The FACSort instrument is a flow cytometer designed for cell sorting. It is capable of rapidly analyzing and sorting cells based on their physical and fluorescent characteristics.
Automatically generated - may contain errors
5 protocols using facsort instrument
1
Lymphocyte Differentiation Assay
2
Cell Population Analysis via Flow Cytometry
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Cultured cells were lifted using enzyme-free cell dissociation buffer (Gibco), resuspended in FACS buffer (PBS supplemented with 5% newborn calf serum) and filtered through 100 μm nylon cell strainers. Samples were fixed in 4% formaldehyde for 10 min at 4 °C and permeabilized in ice cold methanol for 10 min; this fixation/permeabilization step is critical for the cell population distribution analysis. This was followed by staining with anti-CDH1-alexa488 antibody (1:200, Cell Signaling, 3199) for 30 min. Finally, cells were analyzed on a FACSort instrument (BD Biosciences).
3
Cell Cycle Analysis by Flow Cytometry
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Cells were seeded into 6-well plates, at 5×105 cells/well, and incubated for 24 h. Cells were collected by trypsinization and washed once with PBS. The cells were fixed with 70% ethanol overnight at 20°C, washed thoroughly with ice-cold PBS and incubated with a propidium iodide (PI) solution (50 mg/ml RNase A, 0.1% Triton X-100, 0.1 mmol/l EDTA and 50 mg/ml PI) for ≥30 min at 4°C. The samples were evaluated with a FACSort instrument (BD Biosciences) and the data were analysed with ModFit LT 4.0 software (Verity Software House, Inc., Topsham, ME, USA).
4
Testicular Cell Preparation and Analysis
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Testes were scraped and digested with 100 µg/ml collagenase and 100 µg/ml DNase I for 15 min at 37°C, and then filtered through a 40 µm cell strainer (FALCON). The testicular cells were fixed in 70% ethanol and brought to a concentration of 1–2×106 cells/ml in propidium iodide/RNase solution (BD Biosciences 550825). Cells were analyzed by a Becton, Dickinson (Rutherford, NJ) FACSort instrument equipped with an argon laser.
5
Cell Death Analysis by Flow Cytometry
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Cells were washed twice with ice-cold PBS and then resuspended at 1 × 106/mL in a hypotonic fluorochrome solution containing propidium iodide (PI) 50 μg/mL in 0.1% sodium citrate plus 0.03% (v/v) Nonidet P-40. After 1 h of incubation in this solution, the samples were filtered through nylon cloth, 40 μm mesh, and their fluorescence was analyzed as single-parameter frequency histograms using a FACSort instrument (Becton Dickinson, Mountain View, CA, USA). The data were analyzed with CellQuest™ (Becton Dickinson, Mountain View, CA, USA). Cell death was determined by evaluating the percentage of events accumulated in the preG0-G1 position analyzed by flow cytometry.
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