Flow Cytometry Flashcards
(23 cards)
What is Flow Cytometry
Cytometry is a technique for making measurements on cells. Flow Cytometry extends this to measure multiple characteristics of single-cell suspensions as they flow through focused laser beams
What measurements can flow cytometry provide
Cell surface markers: e.g. CD4 (T-cells), CD8 (T-cells), CD19 (B-cells)
Intracellular markers: e.g. cytokines (IL-10, INF-y)
What are the main components of Flow Cytometry
Lasers: Excite fluorochromes
Filters: Direct Specific Wavelengths of light
Detectors (PMTs) : convert light into electronic signals for analysis
What does a photomultiplier tube do?
PMTs amplify photons into electrons, which are then measured as part of the data collection process
What does forward scatter indicate?
It indicates cell size; larger cells scatter more light in the forward direction
What does side scatter indicate
It measures cell granularity and internal complexity, based on light scattered 90° to the laser beam
How are fluorescent dyes used in flow cytometry?
Fluorescent dyes or antibodies bind to specific cell markers and fluoresce when excited by lasers, allowing identification of the cells
What is an example of a dye-marker pairing
CD4-FITC (fluoresces green when excited by a 488 nm laser)
What is a Stokes shift, and why is it important?
The difference between a dyes excitation and emission wavelengths. It allows multiple dyes with different emission spectra to be distinguished simultaneously
What is an example of Stokes shift in dyes used in flow cytometry
FITC (emits green light at 525 nm when excited at 488 nm)
How do two lasers differentiate colors in flow cytometry
Different dyes emit light at unique wavelengths (Stokes shift), allowing multiple fluorochromes to be distinguished even with just two lasers
What are single - parameter histograms used for?
To display the intensity of one marker. They cannot determine if cells co-express multiple markers
What are dual-parameter dot plots used for?
To visualize and identify cells co-expressing two markers
What is gating in flow cytometry?
Gating defines a subset of cells for focused analysis, such as CD4+ T-cells within splenocytes
What are the activation markers for T and B cells?
T: CD25 and CD62L
B: CD25 and CD62L
What mitogens were used in the experiment?
ConA: T - cell mitogen
LPS: B - cell mitogen
How does CD25 expression change upon activation
CD25 expression increases on both T-cells and B-cells upon activation
How does CD62L expression change upon activation
CD62L expression decreases on both T-cells and B-cells upon activation
What are common errors in cell preparation for flow cytometry?
Adding all antibodies to every sample
Failing to label tubes properly
What are common errors in data analysis
Assuming the presence of CD4 means T cell activation (ConA) without comparison to controls or other groups
what are potential applications of flow cytometry and research?
Immunophenotyping
investigating cell signaling pathways
analyzing immune responses
stem cell characterization
what increased on T cells in the conA treated samples?
CD4+CD25+
What increased in LPS treated samples?
CD 25 expression on the cell (CD 19+)
