Immunology- intro. to FACS Flashcards
Name 3 applications for FACS.
Surface immunofluorescence using fluorochromes coupled to antibodies.
Intracytoplasmic fluorescence analysis for detecting cytokine expression.
DNA analysis using fluorescent probes like Hoechst, Dapi, and PI.
Name 3 parameters that can be measured by FACS.
hree parameters that can be measured by FACS are:
Relative size (Forward Scatter—FSC).
Relative granularity (Side Scatter—SSC).
Relative fluorescence intensity using different fluorescent channels (FL1, FL2, FL3, etc.).
Name 3 types of optical filters.
Longpass (LP) filters.
Shortpass (SP) filters.
Bandpass (BP) filters.
Explain the term FACS compensation.
FACS compensation is a process used to correct for spectral overlap in fluorescent signals from different fluorochromes. Since different fluorochromes emit fluorescence at slightly different wavelengths, there can be overlap between the detectors’ emission channels. Compensation involves adjusting the detectors to correct for this overlap, ensuring accurate interpretation of the signals from each fluorochrome.
A patient’s results are as follows:
White Cell Count: 4 x 10^9 cells/liter
Differential White Cell Count:
Neutrophils: 65%
Lymphocytes: 19%
Monocytes: 9%
Eosinophils: 6%
Basophils: 1%
CD4 count: 12% of Lymphocytes
CD8 count: 20% of lymphocytes
Calculate the Absolute count of CD4 and CD8 per microliter.
To calculate the absolute counts, you need to use the white cell count and the percentages of CD4 and CD8 cells in the lymphocyte population.
Absolute CD4 count = White Cell Count × Lymphocyte Percentage × CD4 Percentage
Absolute CD4 count = 4 × 10^9 cells/liter × 19% × 12% = 9.12 × 10^6 cells/µL
Absolute CD8 count = White Cell Count × Lymphocyte Percentage × CD8 Percentage
Absolute CD8 count = 4 × 10^9 cells/liter × 19% × 20% = 15.2 × 10^6 cells/µL
Explain the principle of hydrodynamic focusing in the fluidics system of a flow cytometer.
Hydrodynamic focusing is a technique used in the fluidics system of a flow cytometer to ensure that cells pass through the laser beam in a single file, resulting in accurate measurements. This is achieved by using sheath fluid to create a narrow, central stream of cells, with the sample fluid surrounding it. The sheath pressure determines the velocity of the cells, and the sample pressure controls the rate at which cells are introduced. Hydrodynamic focusing helps to prevent cells from overlapping, ensuring precise analysis.
What is the purpose of optical filters in a flow cytometer’s optical system?
Optical filters in a flow cytometer’s optical system serve to select specific wavelengths of light for detection. Filters act as windows on the spectrum, allowing only certain wavelengths to pass through while blocking others. This is essential for separating the excitation and emission light and minimizing spectral overlap. Filters help to isolate the fluorescence emitted by fluorochromes from the excitation light and other unwanted signals, enabling accurate detection of the desired fluorescence signals.
Describe the process of analog-to-digital conversion in flow cytometry.
Analog-to-digital conversion is a crucial step in flow cytometry’s electronic system. The voltage pulses generated by the photomultiplier tubes (PMTs) in response to the light signals from cells are analog signals. These analog signals need to be converted into digital data that can be analyzed by a computer. Analog-to-digital converters (ADCs) are used for this purpose. ADCs quantize the analog signals into discrete digital values, which can then be assigned to specific channels for analysis, such as height, area, and width of the pulse.
How does fluorescence intensity relate to the process of fluorescence emission in flow cytometry?
Fluorescence intensity is a measure of the amount of light emitted by a fluorochrome molecule after it absorbs energy from an incoming laser beam. When a fluorochrome molecule absorbs energy, it undergoes a transition to an excited state. As the molecule returns to its ground state, it releases the absorbed energy in the form of emitted light. The intensity of this emitted light is proportional to the number of molecules that underwent fluorescence. Measuring fluorescence intensity in specific fluorescent channels allows researchers to quantify specific molecules or markers present in the analyzed cells.
Explain the significance of forward scatter (FSC) and side scatter (SSC) parameters in flow cytometry.
Forward scatter (FSC) and side scatter (SSC) parameters provide valuable information about the physical properties of cells analyzed by flow cytometry. FSC measures the size of the cell and its surface characteristics, such as surface area or roughness. SSC reflects the complexity or granularity of the cell, which is influenced by internal structures and contents. By analyzing FSC and SSC data, researchers can distinguish different cell populations based on size and complexity, aiding in the identification and sorting of cells with distinct characteristics.
