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 : CD19 : CD27 : CD69 010 10 10 10 R1 CD4+ CD4+ : CD178 •CD19-FITC shows a significant drop at the 400:1 dilution Titer_CD19 1,3a,50.fcs Titer_CD27 1,3a,50.fcs Titer_CD69 1,3a,50.fcs CD8+ Event Count: 27707 Event Count: 4991 Event Count: 4966 Titer_CD178 1,3a,50.fcs Event Count: 9775 going from 37.1 to 22.2 % positivity whereas CD27-FITC

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 : CD19 : CD27 : CD69 2 3 4 5 R1 010 10 10 10 respectively. CD4+ CD4+ : CD178 Titer_CD19 1,3a,100.fcs Titer_CD27 1,3a,100.fcs Titer_CD69 1,3a,100.fcs CD8+ Event Count: 27844 Event Count: 5072 Event Count: 4733 Titer_CD178 1,3a,100.fcs Event Count: 10193 •CD69-PE and CD178-PE would be examples of poor antibody

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 : CD19 2 3 4 5 R1 : CD27 : CD69 010 10 10 10 visualization or gating strategy to a different staining approach Titer_CD19 1,3a,200.fcs CD4+ CD4+ : CD178 CD8+ Event Count: 27777 Titer_CD27 1,3a,200.fcs Titer_CD69 1,3a,200.fcs Event Count: 5019 Event Count: 4799 Titer_CD178 1,3a,200.fcs Event Count: 9928 such as an indirect method using biotin in order to amplify a 120 150 weak signal. The myriad of possibilities is so application 600 250 90 specific that it is beyond the scope of this generalized

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 : CD19 : CD27 2 3 4 5 : CD69 010 10 10 10 R1 CD4+ CD4+ : CD178 Titer_CD19 1,3a,400.fcs Titer_CD27 1,3a,400.fcs Titer_CD69 1,3a,400.fcs CD8+ Event Count: 27804 Event Count: 4978 Event Count: 4867 Titer_CD178 1,3a,400.fcs Event Count: 9852

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 : CD19 010 10 10 10 : CD27 : CD69 R1 : CD178 CD4+ CD4+ Titer_CD19 1,3a,800.fcs CD8+ Titer_CD27 1,3a,800.fcs Titer_CD69 1,3a,800.fcs Event Count: 27777 Titer_CD178 1,3a,800.fcs Event Count: 5056 Event Count: 4796 Event Count: 10035

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 : CD27 010 10 10 10 : CD19 : CD69 CD4+ : CD178 R1 CD4+ Titer_CD27 1,3a,1600.fcs CD8+ Titer_CD19 1,3a,1600.fcs Titer_CD69 1,3a,1600.fcs Event Count: 4967 Titer_CD178 1,3a,1600.fcs Event Count: 27731 Event Count: 4825 Event Count: 10134