High‐speed photodamage cell selection using bromodeoxyuridine/Hoechst 33342 photosensitized cell killing

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Abstract
One of the major drawbacks of droplet sorting in a flow cytometer is the relatively low sorting speed. Thus, we have developed an alternative, faster sorting technique: photodamage cell sorting. In a photodamage cell sorter all unwanted cells, as detected with the first, measuring laser, are killed with the second, damaging laser. Thus, the cells need to be photosensitive to the second laser. In addition, a mechanism is needed to switch this laser on and off based on the sorting criteria. In our photodamage cell sorter, the ZAPPER, we use an acousto‐optic crystal to switch the laser beam. Cells are made photosensitive by vital staining with photosensitizers. With cells grown in the presence of 5‐bromo‐2′‐deoxyuridine (BrdUrd) and stained with Hoechst 33342 (H42) at least a 5‐decade cell reduction is accomplished after irradiation with 400 mW UV light. With this system, sorting rates have been achieved of 30,000 cells per second. Due to the selection based on photodynamic killing, this sorting technique is restricted to the selection of viable cells. Photodamage cell sorting seems well suited for isolating viable cells occurring in low percentages or for the sorting of large numbers of cells. Another application can be the sorting of large or fragile cells.