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 ?
This archive was generated by hypermail 2b29 : Wed Apr 03 2002 - 11:51:44 EST