Page 66 - Live-cellanalysis handbook

Live-Cell Analysis Handbook — Third Edition

How Live-Cell Protein Assays Work

Antibody internalization assays Live-cell immunocytochemistry

In the antibody internalization assay, a pH-sensitive antibody To detect cell surface markers, IncuCyte® FabFluor-488, a
labeling reagent, IncuCyte® FabFluor-pH Red, allows the specific Fab conjugated to a continuously fluorescing molecule at
detection of antibodies as they are internalized into the acidic physiological pH, is utilized for antibody labeling. Addition of
conditions of endosomes and lysosomes, enabling real-time, the IncuCyte® FabFluor-antibody complex to living cells enables
kinetic monitoring over the full time-course of internalization. kinetic detection of cell surface markers, making it possible to
track the movement and interaction of cell subsets, reveal the
dynamics of their interactions, and link time- and concentration-
dependent changes in cell surface protein expression to other
morphological and phenotypic parameters.

References

1. Cohen AS, Khalil FK, Welsh EA, Schabath MB, et al. Cell-surface marker discovery for lung cancer. Oncotarget 2017, 8(69): 113373-402.
2. Gundry RL, Boheler KR, Van Eyk JE, and Wollscheid B. A novel role for proteomics in the discovery of cell-surface markers on stem cells:
Scratching the surface. Proteomics Clin. Appl 2008, 2(): 892-903.

3. Macher BA and Yen TY. Proteins at membrane surfaces – a review of approaches. Mol BioSys 2007, 3(): 705-13.
4. Roesli C, Borgia B, Schliemann C, Gunther M, et al. Comparative analysis of the membrane proteome of closely related metastatic and
nonmetastatic tumor cells. Cancer Res 2009, 69(13): 5406-14.

5. Kuhlmann L, Cummins E, Samudio I, and Kislinger T. Cell-surface proteomics for the identification of novel therapeutic targets in cancer. Expert
Review of Proteomics 2018, 15(3): 259-75.
6. Ritchie M, Tchistiakova L, and Scott N. Implications of receptor-mediated endocytosis and intracellular trafficking dynamics in the development
of antibody drug conjugates. mAb 2013, 5(1): 13-21.

7. Beck A, Haeuw JF, Wurch T, Goetsch L, et al. The next generation of antibody-drug conjugates comes of age. Discov Med 2010, 10(53): 329-39.
8. Adams GP and Weiner LM. Monoclonal antibody therapy of cancer. Nat Biotechnol 2005, 23(9): 1147-57.

9. Liao-Chan S, Daine-Matsuoka B, Heald N, Wong T, et al. Quantitative Assessment of Antibody Internalization with Novel Monoclonal Antibodies
against Alexa Fluorophores. PLoS ONE 2015, 10(4): e0124708.
10. Kuo S, Alfano RW, Frankel E and Liu J. Antibody Internalization after Cell Surface Antigen Binding is Critical for Immunotoxin Development.
Bioconjugate Chem 2009, 20(10): 1975-82.

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