Methods

This is a three-color immunophenotyping assay to assess the expression of CD20 on B-cells. We use QuantiBRITE PE beads to establish a fluorescence standard for each experiment. Each of the four bead populations has a calibrated mean number of bound PE molecules/bead. Samples are stained with antibodies that have PE conjugated at a 1:1 ratio. The number of bound PE-con-jugated antibodies/cell in the sample is extrapolated using the PE beads as a standard. Thus, PE molecules bound per cell is equivalent to the number of antibodies bound to the cell. This in turn is equivalent to the number of molecules of target protein in each cell.

m'

FL1-H; CD19 F1TC

1000?

100Q

10"

1000?

100Q

10"

B

i 3.19

39

J

-¡2¿M

1000 10000

100 1000

FL1-H; CD19 F1TC

FL1-H: CD19 FITC

ioooq

W 1000

10"

ioooq

W 1000

10"

c

0.15

" 4.42

2.64, „.y.

■■ ■ .92.6

100 1000 10000

FL1-H: CD19 FITC

100 1000 10000

FL1-H: CD19 FITC

Fig. 1. Dot plots representing examples of CD20 expression in B-cells (CD19+) pre- (A) and post-treatment with rituximab (B,C). The antibody-binding capacity is 1367, 780, and 0 for A, B, and C, respectively.

0 0

Post a comment