Flow cytometry twocolorsinglelaser

As mentioned above, dual staining in flow cytometry presented a real challenge, one which was not met by the fluorescein/rhodamine combination. Dual-laser systems, especially in combination with a dye laser, gave high-quality correlated immunofluorescence data with fluorescein and Texas Red, but at the cost of optical and electronic complexity and at considerable added expense compared with single-laser instruments. PE is now employed routinely together with fluorescein on a single-laser instrument for two-color correlated analysis. The broad excitation spectrum, large extinction coefficient, and high quantum yield of PE make it a reasonable dye to use in combination with fluorescein at 488 nm excitation. As shown by Figure 1 (blue laser fluorescence), the fluorescences are divided by a

560 nm longpass dichroic reflector, suitably filtered, and then the analog signals are compensated to correct for the small overlap of PF. in the fluorescein (FL) channel and the somewhat larger overlap of fluorescein in the PE channel. Data quality compares favorably with two-color staining carried out on a dual-laser system.

Cell suspensions are stained on ice either using directly labeled reagents in a single step or indirectly in multiple incubations separated by washes. Incubations are usually 15-30 min and 2-3 wash steps are probably sufficient to eliminate unbound reagent. Staining medium can be a simple balanced salt solution or even modified cell culture medium and typically contains 5-10% serum (acting as carrier protein) and 0.1 % NaN, (to prevent capping). If the biotin/avidin two-step staining procedure is employed, it is imperative that the staining medium does not contain biotin. Dual staining requires that single-label controls also be included to verify that compensation is correct and that there is no interaction between reagents.

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