CD 19 CD27 CD 69 CD 178
120 600 120 200 90 Antibody Titration for FACS Staining 90 50:1 400 150
60 # Cells # Cells 60 # Cells
# Cells 100 200 30 8.01 25.6 30 55.5 •Histograms show % positive cells for each antigen-fluorophore 40 50 over a range of doubling dilutions from the stock antibody. 0 0 0 2 3 4 5 0 010 10 10 10 0102 103 104 105 0102 103 104 105 2 3 4 5
250 600 120 120 exhibits a significant drop between the 400:1 and 800:1
200 dilutions. 90 90 100:1 400 150 •Being conservative one should move up two dilutions from the # Cells
# Cells 60 # Cells 60 # Cells 100 200 point of the significant drop in % positivity to establish the 5.01 22.3 30 55.1 30 40.5 titration and hence working concentration for a given antibody. 50
0 0 0 Therefore CD19 and CD27 would be put at 100:1 and 200:1 2 3 4 5 010 10 10 10 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 0
600 250 120 120 titration as demonstrated by the lack of a clear peak of 200 positivity and need to be reevaluated. Lack of positivity in 90 200:1 400 90 150 these cases could be due to numerous reasons. Anything # Cells # Cells 60 # Cells 60 # Cells from having a bad antibody to needing an infected animal in 200 100 2.38 20.1 30 53.9 30 37.1 order to see a particular rare marker could explain these 50 0 results. Solutions may involve anything from a different 2 3 4 5 0 0 010 10 10 10 0 0102 103 104 105 0102 103 104 105
100 200 explanation of antibody titration methodology. 400:1 400 60
# Cells # Cells 150 # Cells # Cells
200 50 100 30 0.92 17.4 47.7 22.2 50
0 0 0 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 2 3 4 5 0 010 10 10 10
300 150 600 100
80 100 200 800:1 400 60 # Cells # Cells # Cells # Cells Working dilution for antibody. 40 200 50 100 14.5 0.54 32.3 7.87 20
0 0 0 2 3 4 5 0 010 10 10 10 2 3 4 5 2 3 4 5 010 10 10 10 010 10 10 10 2 3 4 5
120 300 150
600 90
200 100 1600:1 400 60 # Cells # Cells # Cells # Cells
100 12.6 200 30 50 0.21 6.18 1.71
0 0 0 2 3 4 5 0 2 3 4 5 010 10 10 10 2 3 4 5 010 10 10 10 010 10 10 10 2 3 4 5