antibody concentration is less than 1 mg ml the conjugation will probably be sub optimal if necessary dilute the antibody to a concentration of 4 mg ml dissolve 10 mgs the entire contents of one vial no need to weigh of fitc molecular probes in anhydrous dmso immediately before use add fitc to give a ratio of 40 80 u03bcg per mg of antibody mix immediately wrap the tube in foil incubate and rotate at room temperature for one hour remove the unreacted fitc and exchange the antibody into 500 mm carbonate ph 9 5 by gel filtration or dialysis determine f p and protein concentration by measuring the absorbance at 280 and 495 nm igg 1 mg ml has an a 280 of 1 4 mw 150 000 igm 1 mg ml has an a 280 of 1 2 mw 900 000 fluorescein 1 mm has an a 495 of 68 and an a 280 of 11 8 f p values of 3 10 are probably optimal for any particular igg a bone marrow sample is obtained from an all patient and leukocytes are isolated according to standard procedures leukocytes are labeled with two cell surface markers to define a leukocyte subset via immunophenotypic characteristics fitc conjugated monoclonal anti human cd19 fitc cd19 and pe conjugated monoclonal anti human cd10 pe cd10 were used cells are then fixed according to standard procedures e g in 1 paraformaldehyde to preserve the integrity of the cell and its content the cell membrane is permeabilized using a detergent such as saponin to make the cell interior accessible.