**2.4 Propidium iodide**

Propidium iodide is an analogue of ethidium bromide. It is an exclusion dye that binds to short non-specific double stranded RNA or DNA at excitation wavelength of 525 nm and an emission wavelength of 491–495 nm to produce red or orange fluorescence [44]. Propidium iodide is impermeable to the plasma membrane in living cells however it easily permeates compromised plasma membrane [45–47]. Once inside the cell, the dye binds to DNA, which leads to a 30-fold increase in fluorescence shifting the excitation maximum by 30–40 nm up to 525 nm to the red and emission maximum by 15 nm to the blue [44–46]. Thus, propidium iodide provides an objective number of dead or necrotic cells in a cytotoxicity assay when observed with a fluorescent microscope or flow cytometer (**Figure 4**). Therefore, assessing cytotoxicity with propidium iodide is easy, cost-effective and aids in measuring a large set of samples using automated flow cytometer.

In addition to acridine orange, propidium iodide is often co-stained with annexin V [12, 38, 41]. Annexin V binds to phosphatidylserine (PS) exposed on the outer membrane of necrotic cell surface. PS is usually situated in the inner membrane leaflet in viable cells and only translocate to the outer membrane leaflet in these cells upon induction of apoptosis [12, 48]. Co-staining propidium iodide with annexin V offers the ability to simultaneously detect and measure viable and non-viable cells. The cells are grouped into four groups: (a) viable cells (annexin V-negative/propidium iodide-negative), (b) necrotic cells (annexin V-negative/propidium iodide-positive), (c) early apoptotic (annexin V-positive/propidium iodide-negative), (d) late apoptotic (annexin V-positive/propidium iodide-negative) [3, 10, 12, 49]. Therefore, propidium iodide co-staining with annexin V or acridine orange provides reliable, reproducible results and distinguishes subpopulation of apoptotic cells with accuracy.

However, in some cases, late apoptosis can be characterised by some loss of membrane integrity therefore flow cytometric analysis of cytotoxicity using propidium iodide and annexin V cannot differentiate between late apoptosis and necrosis, which can both be annexin V-positive and propidium iodide-positive. In addition, apoptotic cells stained with propidium iodide present different hypodiploid peaks and sizes on the red fluorescent channels, which could be representative of the sub G1 (debris) in the sample [46, 49]. Therefore, despite propidium iodide being a universal cell death indicator, results can be skewed by nuclear material in solution although this

#### **Figure 4.**

*Cells treated with 20 μg/mL of PI/FACS buffer for 10 min on Ice and analysed on the flow cytometer. Exclude debris using (P1 left panel) then isolate non-viable cells on the PE-A vs. FSC-A using P2 (right panel).*

*Trypan Blue Exclusion Assay, Neutral Red, Acridine Orange and Propidium Iodide DOI: http://dx.doi.org/10.5772/intechopen.105699*

can be avoided by excluding the debris by size in the forward and side scatter plots or using RNAse A to digest RNA. Propidium binding to the DNA can be affected by the chromosome structure and this can be exploited to study the effects of drugs and xenobiotics on cell cycle [46, 49]. This is because propidium shares similar properties to some chemotherapy agents such as mitoxantrone, which can mask its reading and lead to underestimation of toxic effects [50, 51].
