>I have a researcher who would like to utilize PKH26 as a tracking dye >for rabbit rbc's survival studies. I have gotten reasonable separation >of positives and negatives using a "PE" filter setup (555DLP beam >splitter and 580/30 band pass) with 488 excitation on a MoFlo. The >researcher is worried about cell toxicity of the PKH26 and wants to use >as little as possible in his staining procedure. In an attempt to >maximize fluorescence intensity, I've tried to "tune" the dye head laser >(INNOVA 70) This is confusing here. Are you running an I-70 argon laser, or are you using the argon laser to pump a tunable dye laser? If you are using the tunable dye laser that is commonly used in flow cytometry, it will not give much output below 575 nm. Thus, you will see scattered laser light in your PE passband. If you are using an I-70, you can tune it to its green line (514 nm). PKH 26 excitation curve has a shoulder there which is 60% of the 551 nm maximum, so you should get results here that are probably better than you can with 488 nm excitation. Whether you use 488 nm excitation or 514 nm excitation, you can optimize you detection filters. A 540 LP filter will collect more PKH 26 signal than the 580/30. If you decide to try this, you should configure your optics so that there isn't a beamsplitter cutting into this detection zone. No matter which laser line you use, varying the laser power to optimize the signal to background is worth looking into. Lastly, Sigma makes another dye PKH 2, similar to PKH 26, but well-matched to the 488 nm line of an I-70. I have not used this dye, so I cannot say how well it performs. Marty Bigos Gladstone Flow Core > on the MoFlo to maximum excitation for PKH26 (551 nm) but >seem to overwhelm the detection PMT with its 580/30 band pass. Any >suggestions or advice on filter setup or use of PKH26 in this scenario >is much appreciated.
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