DCFDA+(5-(and-6)-carboxy-2′,7′-dichlorofluorescein+diacetate)

__** DCFDA **__, an indicator of the presence of oxidation. __** Introduction: **__

Commonly known as DCFDA, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein diacetate, is commonly used as an indicator of oxidative stress, oxidative stress can be classified as a situation where oxidation is not taking place. Typically DCFDA is introduced into target cells through a small amount of aqueous solution (Jilani, Lupescu, Zbidah, Shaik, Lang, 2012). After the DCFDA is introduced to the cell, it rapidly diffuses through the cell membrane as a colorless probe. It is only once the two acetate groups have been cleaved by the esterases inside the cell that the DCFDA becomes fluorescently visible. A valuable property of DCFDA is that once it has been cleaved inside the cell it cannot exit the cell and is therefore an indicator that can be used for a greater length of time (Pogue et al., 2012).

__** Purpose: **__

The purpose of visualizing cells using DCFDA is to detect oxidative stress since DCFDA is only visible when the cell is suffering from oxidative stress.

__** Origins: **__

What I can conclude about the use of DCFDA is that it is a relatively new technique that has only become widely documented in the last two years. Little information was available based on the discovery and/ or origins of this technique therefore all that I can do is speculate on its details. I speculate that it was discovered around the need for an indicator to detect oxidation probably based on a need to research cell death sue to oxidative stress.

__** Examples of different uses and forms: **__

The first article utilizing DCFDA was to detect the suicidal death of erythrocytes upon exposure to Withaferin A, this compound counteracts malignancy through oxidative stress, altered gene expression, and depolarized mitochondria leading to apoptosis. Thus using DCFDA to detect oxidative stress in cells exposed to Withafrein A was essential to the conclusion of this experiment.

The second article utilizing DCFDA was to detect the presence of ROS, reactive oxygen species (linked to premature cell aging), caused by metal-sulfates in human brain cells. ROS are naturally occurring in human cells, however an excess of ROS generation overwhelms the cells anti-oxidant defense and in turn leads to oxidative stress. This, again, is detected through the use of the indicator DCFDA. The interesting point in this article is that a different form of DCFDA was used this one was a Carboxy-DCFDA.

The third article utilizing DCFDA was again to detect the presence of ROS, this time in dermal dendritic cells of a mouse after exposure to an allergen. The presence of ROS and in turn the level of oxidative stress was detected through the use of the indicator DCFDA. This experiment used a Hydroxy-DCFDA.

__** References: **__

Jilani, K., Lupescu, A., Zbidah, M., Shaik, N., Lang, F. (2012). Withaferin A-stimulated Ca2+ entry, ceramide formation and suicidal death of erythrocytes. //Toxicology in Vitro.// Retrieved October 13, 2012, from ScienceDirect database.

Miyazawa, M., Takashima, A. (2012). Development and validation of a new //in vitro // assay designed to measure contact allergen-triggered oxidative stress in dendritic cells. //Journal of Dermatological Studies, 68//(2). 73-81. Retrieved October 10, 2012 from ScienceDirect database.

Pogue, A. I., Jones, B. M., Bhattacharjee, S., Percy, M. E., Zhao, Y., Lukiw, W. J. (2012). Metal- Sulfate induced generation of ROS in human brain cells: detection using an isomeric mixture of 5- and 6-Carboxy-2′,7′-Dichlorofluorescein Diacetate (Carboxy-DCFDA) as a cell permeant tracer. //International Journal of Molecular Biology, 13//(8), 9615-9626. Retrieved October 12, 2012 from PubMed database.