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To demonstrate the multiplexing

alone or when we mixed them. This result

capabilities of this novel pH-sensitive dye on

shows that multiplexing positive and negative

the iQue® platform, we used Ramos and Raji

cell lines does not interfere with the antibody

cells stained with two intensities of violet

internalization assay.

encoding dye, combined with unstained

Compared to performing a series of singleplex

Jurkat cells. We incubated this for three hours

assays, a multiplexed assay approach enables

with a serial dilution of dye-conjugated

you to analyze multiple readouts (internalization,

specificity antibodies (isotype-matched

viability, cell type) from a single well, decreasing

anti-CD3 as a T cell marker, anti-CD19, anti-

the number of tests needed to perform a

CD20, anti-CD22, or anti-CD79b as B cell

comprehensive functional characterization of the

markers, anti-CD71 as a positive control, and

Ab candidate.

IgG as a negative control), then added a cell
membrane integrity dye before acquiring data

Full Concentration Profiling with

from the plate. Using this strategy, we were

Live-Cell Imaging and Analysis

able to identify viable cells, then spectrally

A pH-sensitive dye-coupled antibody fragment

separate Ramos, Raji, and Jurkat cells.

designed according to the same principle as

We then assessed antibody internalization

that used above (Figure 2) can also be optimized

for each cell line. We generated series dilution

and used in other instruments. For instance,

curves for the specificity markers of each cell

using this pH-sensitive reagent in a real-time,

type (Figure 4A). As expected, Jurkat cells showed

live-cell imaging system such as the Incucyte®

internalization of anti-CD3, but not anti-CD19

Live-Cell Analysis System, allows visualization and

or anti-CD22, whereas the Raji cells internalized

automatic quantification of the full time-course of

anti-CD19 and anti-CD22, but not anti-CD3. Only

ABI. This combination of reagent and platform thus

Ramos cells showed a concentration-dependent

provides a simple method for directly profiling and

increase in internalization of anti-CD79b, an ADC

comparing ABI for a large number of antibodies

drug target for non-Hodgkin's lymphoma. In the

(10-100s at a time in a miniaturized format).

three-hour assay time frame, we did not observe

To demonstrate the power of the pH-sensitive

anti-CD20 internalization, but we did see an

dye approach for high-throughput antibody

increase in the two B cell lines by 24 hours (data

internalization assays in a real-time, live-cell

not shown). Importantly, we saw little difference

analysis system, we performed a head-to-head

when we assessed the cells for internalization

comparison of the internalization properties of

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