At 09:47 PM 1/31/98 +0000, you wrote: > >Today we tried to run four fluorochrome surface markers using the B-D >multitest cd3/cd8/cd45/cd4 (fitc/pe/perCP/apc) and found an interesting >problem with our fluorescent data plots. we use dako's UTILYSE as our >no-wash whole blood reagents. We have a facstar + multi laser instrument >and used the he -ne laser for excitation of the apc and argon for the >others. Our ssc vs apc fluorescence data plots showed a definite non >specific fluorescence in the neutrophils that was as bright or brighter >than the cd4 apc pos lymphocytes. Repeating the test using B-D facslyse >as the "no-wash" eliminated the neutrophil fluorescence in the ssc vs >apc plots. >Dako's reagent set consists of a reagent 'A' which seems to be a fixative >and a reagent 'B' which seems to be the rbc lysing reagent. The reagent >'B' has a bluish color to it (apc is blue!) and is this the culprit >causing our neutrophil >fluorescence? Are the neurophils somehow picking this up from the reagent >B and not the other cells?Comments or discussion ? Any one else have >this problem ? > > > Hi kstaplet, O.K, this is a crazy idea but it might just work..... if as you suspect, it is reagent B which may be contributing to the non specific fluorescence in your neutrophils you might try the following; Take a small quantity of reagent B (say about 5-10ml). Add an excess of Activated Charcoal (Sigma C-5260). Agitate the above for a couple of minutes at room temp. Filter through a 0.47um syringe filter. Hey presto... no more non sp. fluorescence (maybe) If you try the above, please let me know how you get on (ie if it works!) Regards Arnold.
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