By Paul Graham.
ANSI universal Lisp ideas.
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Cytofix Buffer; Cat. No. 554655) for 30 min at 4 °C, and finally washed, resuspended in FACS buffer, and stored at 4 °C (protected from light). For each sample, 1,000,000 total events were acquired on the flow cytometer (LSRFortesa; BD Biosciences Systems San Jose, CA) equipped for the detection of 18 fluorescent parameters. 1 (TreeStar, Ashland, OR). To define positive and 30 Iulia Popescu et al. negative populations, we employed fluorescence minus one controls for each fluorophore used in this study, when initially developing staining protocols.
Following the incubation step add 2 ml staining buffer to each tube and pellet by centrifugation at 200 × g for 10 min. The important steps for flow cytometry assay are the following: 1. Configure your instrument. 2. Characterize your instrument. 3. Design your panel. 4. Optimize settings for your panel. 5. Run appropriate controls. 6. QC your data [8–10]. ). Acquisition of the data is mediated by a computer physically connected to the flow cytometer, and the software which handles the digital interface with the cytometer.
Similar results were obtained when we analyzed CD8+IFN-γ+ effectors for CD45RA expression, as seen in Fig. 3. We should also mention that, as an additional control, we also assessed the memory 34 Iulia Popescu et al. marker CCR7 and found that the vast majority of CMV-specific effectors were CCR7− in both the blood both during and following acute infection (data not shown). Together, these data show that CMV-specific CD8+ T cells from the blood transition from a CD45RA− phenotype to a CD45RA+ phenotype as viremia/acute infection resolves, indicating distinct phenotypic differences between CMV-specific CD8+ effector memory T cells in the lung and PBMC during chronic infection .