Why is FITC used for Excitation and Emission in Flow Cytometry?

In flow cytometry, laser light is usually used to excite the fluorochromes. These lasers produce light in the UV and/or visible range. Fluorochromes are selected based on their abilities to fluoresce with the wavelengths of light produced by the lasers. The electrons of a fluorochrome can be excited by a range of wavelengths of light. This optimal wavelength is called the excitation peak. Similarly, the light produced by fluorochromes has a range of wavelengths and this optimal wavelength is called Emission Peak.

At what wavelength FITC is measured in FACS?

Knowing the excitation 495 nm and emission 520 nm wavelengths of fluorescent compounds makes it possible to select combinations of fluorochromes that will work together optimally on a specific flow cytometer with specific lasers.

FITC Labeled Antibody

Excitation Peak (nm) 495
Emission Peak (nm) 520
Laser Wavelength (nm) 488

Fluorescence Measurement

Fluorescence measurements taken at different wavelengths can provide quantitative and qualitative data about fluorochrome-labeled CDs, Cluster of Differentiation, cell surface receptors or intracellular molecules. Flow cytometers use separate fluorescence (FL-) channels to detect light emitted. The number of detectors will vary according to the machine and its manufacturer. Detectors are either silicon photodiodes or photomultiplier tubes (PMTs). Silicon photodiodes are usually used to measure forward scatter when the signal is strong. PMTs are more sensitive instruments and are ideal for scatter and fluorescence readings.
The specificity of detection is controlled by optical filters, which block certain wavelengths while transmitting (passing) others. There are three major filter types. ‘Long pass’ filters allow through light above a cut-off wavelength, ‘short pass’ permit light below a cut-off wavelength and ‘band pass’ transmit light within a specified narrow range of wavelengths (termed a band width). All these filters block light by absorption.
As the fluorescing cell passes through the laser beam, it creates a peak or pulse of photon emission over time. These are detected by the PMT and converted to a voltage pulse, known as an event. The total pulse height and area is measured by the flow cytometer. The measured voltage pulse area will correlate directly to the intensity of fluorescence for that event. The pulse area is calculated by adding the height values for each time slice of the pulse, determined by the speed of the analog to digital converter (ADC), which is 10 MHz (i.e. 10 million per second or 10 per microsecond).
To represent the data, histograms and dot-plots are used and both provide different advantages for flow cytometry data analysis.

So, how to choose which one is best represent your data?

Histogram
• Fast to read and easy to understand.
• Most useful when only one parameter (e.g. intensity from a single fluorescent channel) is important.
• Usual representation includes the intensity of a single channel (horizontal axis) vs number of detected events (vertical axis).
• Multiple overlaid histograms can be used to compare a single parameter from two different sample populations (e.g. experimental vs. control).
Dot plot
• Most useful when you need to compare multiparametric data (e.g. Intensity of side-scatter vs forward-scatter channels).
• Can be two- or three-dimensional
• Intensity of each channel is represented on its own axis.
• Each distinct event is represented at a single dot.
• A more complex, more illustrative representation of data.

How many types CD4 Antibody are there?

Genprice sells 20 anti-CD4 mouse monoclonal antibodies. Highly expressed in T-helper cells. The presence of CD4 is a hallmark of T-helper cells which are specialized in the activation and growth of cytotoxic T-cells, regulation of B cells, or activation of phagocytes. CD4 is also present in other immune cells such as macrophages, monocytes, dendritic cells or NK cells.

How many types of CD8 alpha Antibody are sold by Gentaur?

There are 16 anti-CD8 alpha mouse monoclonal antibodies. The CD8 glycoprotein is expressed by thymocytes, mature T cells and natural killer (NK) cells and has been implicated in the recognition of monomorphic determinants on major histocompatibility complex (MHC) Class I antigens, and in signal transduction during the course of T-cell activation. Both human and rodent CD8 antigens are comprised of two distinct polypeptide chains, alpha and beta. The Ig domains of CD8 alpha are involved in controlling the ability of CD8 to be expressed. Less frequently, CD8 can be expressed as a CD8A homodimer. A subset of natural killer cells, memory T-cells, intraepithelial lymphocytes, monocytes and dendritic cells expresses CD8A homo-dimers. Expressed at the cell surface of plasmacytoid dendritic cells upon herpes simplex virus-1 stimulation.

Djoumana Ounas

Djoumana Ounas

Pharmacist Assistant Professor in Analytical Chemisty. Consultance Expertise and Reglatory Biotech Support.

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