University of Vermont

College of Medicine

Flow Cytometry and Cell Sorting (FCCS) Facility

Functional Assays

Functional Assays


Cell of the immune system continuously undergo expansion and differentiation during development and immune responses (importantly antigen-specific responses). The ability to characterize the proliferative capacity and susbsequent expansion of immune cells is critical to understand an immune phenomenon. There are different protocols for determining cell division (in vivo and in vitro).

  • Carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)-. It is cell membrane permeable and it is cleaved by intracellular estearase enzymes to form an amine-reactive product (CFSE) which is stabilized by crosslinking to intracellular and extracellular proteins (lysine residues). If a cell divides, the dye is divided equally between daughter cells, resulting in a reduction of fluorescence as division progresses (see figure). This technique allow the identification up to 8 successive cell divisions. CFSE is excited by 488 nm laser with a peak emission of 520 nm (530/30 Bandpass). Protocol.
  • CellTrace™ Violet for cell proliferation (LifeTech/Invitrogen): For in vitro and in vivo labeling of cells to trace multiple generations using by dilution. Excitation at 405 nm, 450/50 Bandpass.
  • Violet Proliferation Dye 450 (BD Biosciences): Excitation 405 nm, 450/50 Bandpass.

Calcium flux

In their resting state, eukaryotic cells maintain an internal Ca++ concentration that is far lower than that of the extracellular environment. Elevation of intracellular calcium ions is one of the most rapid cellular responses and used as an indicator of cellular activation. Flow cytometry can be used to measure the concentration of intracellular free calcium iones (figure) when an appropriate sensor such a s Indo-1 AM is used. The emission spectrum of Indo-1 changes from blue to violet upon binding to Ca++. The ratio of violet to blue fluorescence is independent of the amount of dye within the cell. The increase in the ratio over time reflects the increase in intracellular Ca++ concentrations.

How to Set Up a Calcium Flux Experiment (Flow Cytometry)? Click here.

Reactive Oxygen Species (ROS)

ROS are chemical reactive molecules containing oxygen (including oxygen and peroxide) produce by normal metabolism of oxygen. Changes in ROS levels are associated with normal physiological processes such as cell proliferation, phagocytosis, apoptosis, and also during inflammation, cancer, and environmental stress (heat exposure, starvation, UV, etc).

ROS can be detected by flow cytometry using several dyes. Among them, the cell permeant 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA, LifeTech/Molecular Probes) is a reduced form of fluorescein used as an indicator for ROS in cells. Upon cleavage of the acetate group by intracellular estearases and oxidation, the non-fluorescent H2DCFDA is converted to the highly fluorescent 2',7'-dichlorofluorescein (DCF). Molecular Probes has different version of this indicator (Carboxy-H2DCFDA and di-acetoxymethyl ester) with better cellular retention. See figure.

In addition, MitoSox Red™ (LifeTech/Molecular Probes) is specifically designed to measure the production of superoxide (but not other ROS or reactive nitrogen species, RNS) by mitochondria.

Last modified January 24 2014 02:48 PM