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Multiplexed Viability/Encoding Antibody Internalization Reagent Workflow
Violet Encoding
Dye (V/Blue)

Cell Preparation

Label test antibodies with
Antibody Internalization
Reagent and prepare dilutions.


Encode cell populations with
Violet Encoding Dye and plate
encoded cells.

Add labeled antibody
and Membrane Integrity
Dye (B/Green).
Incubate two hours.

Acquire data
on iQue3 VBR.

Figure 3: The assay consists of 3 components, each 10 µL: labeled test antibody, cells (encoded or not), and Cell Membrane
Integrity Dye (B/Green). Each component is prepared at 3X before addition for a final concentration of 1X and an assay volume
of 30 µL.

a comprehensive, integrated solution for rapid

characteristics within the same workflow. For

profiling of antibody internalization and

instance, you can measure cell viability using a

other critical antibody attributes using small

membrane integrity dye to assess general cell

sample volumes in 96- or even 384-well plate

health, as well as cell death due to cargo delivery,

formats (Riedl et al., 2016). Much of the data

when optimizing ADCs. Or you can characterize

acquisition and analysis, including generation

cell specificity using encoding dyes (for cell lines)

of serial dilution curves and EC50 calculations, is

or directly conjugated fluorescent antibodies

automated with the help of advanced software

(for complex cell models with a variety of cell

packages, for example, as included in the iQue®

types). Other reagents, such as those for assessing

advanced flow cytometry platform.

cytokine release, are also available for more
detailed antibody assessments on the sample.

Functional Profiling for

Therefore, analysis with an advanced flow

Comprehensive Cell and Antibody

cytometry platform can be optimized to deliver

Characterization Early in the

rich content very quickly while only using a low

Process with Multiplex Analysis

sample volume.

Combining the above-described pH-sensitive dye

Sartorius produces such a pH-sensitive

with other reagents in one assay on an advanced

reagent for use on our iQue® advanced

flow cytometry platform enables simultaneous

flow cytometry platform. The combination

analysis of a variety of cell and antibody

of non-perturbing and validated reagents

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