What is FITC flow cytometry?
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. FITC has excitation and emission spectrum peak wavelengths of approximately 495 nm and 519 nm, giving it a green color. Like most fluorochromes, it is prone to photobleaching.
What is the principle of flow cytometer?
Flow cytometry (FCM) is a technique which enables rapid analysis of statistically significant number of cells at single cell level. The main principle of this technique is based on scattering of light and emission of fluorescence which occur when a laser beam hits the cells moving in a directed fluid stream.
How do you measure FITC?
The easiest way to determine the FITC to Antibody (F/A) ration, meaning how many FITC molecules you have on average on one molecule, or antibody concentration you can use a spectrometer (NanoDrop for example) and measure the absorbance at the wavelengths 280 and 495 nm.
What is Fluorochrome in flow cytometry?
Fluorochromes used in flow cytometry are essentially those that can attach in some way to biologically significant molecules and are excitable by the lasers commonly found on commercial flow cytometers.
What are the three main systems in a flow cytometer?
A flow cytometer is made up of three main systems: fluidics, optics, and electronics. The fluidics system transports particles in a stream to the laser beam for interrogation.
How do you make a FITC?
Dissolve FITC in anhydrous DMSO at 1 mg/ml concentration. This should be prepared fresh for each labelling reaction. Add 5 μl of the dye to the dialysed protein (5 ml) every 30 min to get a final concentration of 100 ng FITC/1 μg protein. Perform this step by gently rocking the protein solution at 4°C for 12 h.
What is FITC and PE in flow cytometry?
The FITC / PE Compensation Standard is to be used in conjunction with hardware or software to remove spectral overlap from fluorochromes into secondary fluorescence detectors of a flow cytometer. The FITC/PE Compensation Standard is a mixture of 4 populations of microspheres: FITC, PE, FITC/PE, and AutoFluor™.
Is af488 the same as FITC?
Alexa Fluor 488 (Ex-Max 495 nm/Em-Max 519 nm): have nearly identical emission and excitation maxima as FITC. In addition, Alexa Flour 488 is pH insensitive (over a broad pH range) and tends to be brighter compared to FITC on most instruments.
What is fluorochrome in flow cytometry?
What is the role of FITC in flow cytometry?
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure.
How are FS and SS used in flow cytometry?
Cells or particles passing through the beam scatter light, which is detected as FS and SS. FS correlates with cell size and SS is proportional to the granularity of the cells. In this manner, cell populations can often be distinguished based on differences in their size and granularity alone.
What is the purpose of a flow cytometry instrument?
Flow cytometry (FCM) is a technique used to detect and measure physical and chemical characteristics of a population of cells or particles. In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument.
How is fluorescein isothiocyanate used in flow cytometry?
Fluorescein isothiocyanate (FITC) is a derivative of fluorescein used in wide-ranging applications including flow cytometry. First described in 1942, FITC is the original fluorescein molecule functionalized with an isothiocyanate reactive group (-N=C=S), replacing a hydrogen atom on the bottom ring of the structure.