A Comparison of the Sensitivity of Photodamage Assays in Rat Basophilic Leukemia Cells¶

Abstract

Many aspects of cellular function or physiology can be used to indicate the level of damage resulting from the application of potentially deleterious agents such as drugs, solvents or even light. The dose required to reach a specific biological endpoint will necessarily depend on the characteristics of the damage induced by the agent. By using multiple biological probes, it is possible to get a more complete description of the type of damage induced. Photodamage was induced in rat basophilic leukemia cells by either 254-nm UVC light exposure or rose bengal photosensitization. Damage was measured by three quantitative assays employing fluorescent probes: calcein, to measure nonspecific esterase activity, propidium iodide (PI), to measure loss of plasma membrane integrity, rhodamine 123 (R123) to measure mitochondrial depolarization, and the incorporation of 5'-bromodeoxyuridine (BrdU), to measure the progress of cell replication. BrdU incorporation was found to be the most sensitive indicator for both forms of photodamage. For UVC photodamage, the BrdU assay was 330 times more sensitive than the other two assays. For rose bengal photosensitization, the BrdU assay was 48 or 62 times more sensitive than either the R123 or calcein/PI assays, respectively.

PEG-Attached PAMAM Dendrimers Encapsulating Gold Nanoparticles: Growing Gold Nanoparticles in the Dendrimers for Improvement of Their Photothermal Properties

“…Then, green light (λ = 532 nm) was irradiated to the cells to the 3-mm-diameter area for 15 min using a Nd:YVO4 laser. After the irradiation, the live and dead cells were stained, respectively, with calcein-AM (green) and PI (red) . Figure A portrays the calcein fluorescence of the cells treated with the PEG-attached dendrimers encapsulating ungrown Au NPs.…”

“…Then, green light (λ ) 532 nm) was irradiated to the cells to the 3-mm-diameter area for 15 min using a Nd:YVO4 laser. After the irradiation, the live and dead cells were stained, respectively, with calcein-AM (green) and PI (red) (30)…”

Photobleaching of Reduced Nicotinamide Adenine Dinucleotide and the Development of Highly Fluorescent Lesions in Rat Basophilic Leukemia Cells During Multiphoton Microscopy

“…Preliminary experiments were also conducted to determine the immediate biological effect of HFL formation on the living cell. Cells that had developed HFLs were incubated with propidium iodide (PI) and calcein‐AM following a standard protocol (25). The cellular uptake of these fluorescent probes provides a well‐known measure of cell viability.…”

“…Previously we have measured a reduction of DNA synthesis in RBL cells imaged under the same conditions used in this current study (20). Changes in the level of DNA synthesis provide a very sensitive indicator of sublethal photodamage because this occurs at much lower photodynamic doses than that required to adversely affect membrane integrity or esterase activity or cause mitochondrial distress (25). It remains to be seen whether there is a direct connection between NAD(P)H photobleaching and the interruption of DNA synthesis and possibly other forms of cellular damage associated with two‐photon imaging that have been reported.…”

Dually functionalized dendrimers by temperature-sensitive surface modification and gold nanoparticle loading for biomedical application

“…8). The temperature-sensitive PDEG-G5-Au 55 dendrimer was added to HeLa cells, a cervical cancer cell line, and incubated at 37 C for 2 h. Aer washing and staining with calcein-AM and propidium iodide (PI), which respectively show live and dead cells, 48 the cells only slightly displayed PI uorescence, demonstrating that the dendrimer did not induce cellular death under this condition without laser irradiation (Fig. 8A).…”