Herein, an advanced bioconjugation technique to synthesize hybrid polymer-antibody nanoprobes tailored for fluorescent cell barcoding in flow cytometry-based immunophenotyping of leukocytes is applied. A novel approach of attachment combining two fluorescent dyes on the copolymer precursor and its conjugation to antibody is employed to synthesize barcoded nanoprobes of antibody polymer dyes allowing up to six nanoprobes to be resolved in two-dimensional cytometry analysis. The major advantage of these nanoprobes is the construct design in which the selected antibody is labeled with an advanced copolymer bearing two types of fluorophores in different molar ratios. The cells after antibody recognition and binding to the target antigen have a characteristic double fluorescence signal for each nanoprobe providing a unique position on the dot plot, thus allowing antibody-based barcoding of cellular samples in flow cytometry assays. This technique is valuable for cellular assays that require low intersample variability and is demonstrated by the live cell barcoding of clinical samples with B cell abnormalities. In total, the samples from six various donors were successfully barcoded using only two detection channels. This barcoding of clinical samples enables sample preparation and measurement in a single tube.

CLIP Department of Paediatric Haematology and Oncology 2nd Faculty of Medicine Charles University and University Hospital Motol 5 Úvalu 84 Prague 150 06 Czech Republic

Department of Biomedical Polymers Institute of Macromolecular Chemistry CAS Heyrovského nám 2 Prague 162 00 Czech Republic

R and D division 1 T A Intertact s r o Černokostelecká 143 Prague 108 00 Czech Republic

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S.-R. Wang, N.a Zhong, X.-M. Zhang, Z.-B. Zhao, R. Balderas, L. Li, Z.-X. Lian, Cytometry, Part A 2021, 99, 273.

L. M. Park, J. Lannigan, M. C. Jaimes, Cytometry, Part A 2020, 97, 1044.

B. Akkaya, P. Miozzo, A. H. Holstein, E. M. Shevach, S. K. Pierce, M. Akkaya, J. Immunol. 2016, 197, 2027.

P. O. Krutzik, G. P. Nolan, Nat. Methods 2006, 3, 361.

J. ? A. Lee, A. Hung, S. Mardyani, A. Rhee, J. Klostranec, Y. Mu, D. Li, W. C. W. Chan, Adv. Mater. 2007, 19, 3113.

C.-T. Kuo, H.-S. Peng, Y.u Rong, J. Yu, W. Sun, B. Fujimoto, D. T. Chiu, Anal. Chem. 2017, 89, 6232.

H. E. Mei, M. D. Leipold, A. R. Schulz, C. Chester, H. T. Maecker, J. Immunol. 2015, 194, 2022.

F. J. Hartmann, E. F. Simonds, S. C. Bendall, Sci. Rep. 2018, 8, 10770.

J. P. Vial, F. Lacombe, Methods Cell Biol 2001, 64, 343.

F. Garrido, T. Cabrera, A. Concha, S. Glew, F. Ruiz-Cabello, P. L. Stern, Immunol Today 1993, 14, 491.

F. Garrido, F. Ruiz-Cabello, T. Cabrera, J. J. Pérez-Villar, M. López-Botet, M. Duggan-Keen, P. L. Stern, Immunol Today 1997, 18, 89.

K. Masuda, A. Hiraki, N. Fujii, T. Watanabe, M. Tanaka, K. Matsue, Y. Ogama, M. Ouchida, K. Shimizu, K. Ikeda, M. Tanimoto, Cancer Sci. 2007, 98, 102.

R. E. Brouwer, P. Van Der Heiden, G. M. T. Schreuder, A. Mulder, G. Datema, J. D. H. Anholts, R. Willemze, F. H. J. Claas, J. H. F. Falkenburg, Hum Immunol 2002, 63, 200.

D. Kužílková, J. Puñet-Ortiz, P. M. Aui, J. Fernández, K. Fišer, P. Engel, M. C. van Zelm, T. Kalina, Front Immunol 2022, 13, 827898.

F. Junker, P. Camillo Teixeira, Cytometry, Part A 2022, 101, 909.

Á. Szabó, T. Szendi-Szatmári, L. Ujlaky-Nagy, I. Rádi, G. Vereb, J. Szöllosi, P. Nagy, Biophys. J. 2018, 114, 688.

D. Majonis, O. Ornatsky, D. Weinrich, M. A. Winnik, Biomacromolecules 2013, 14, 1503.

K. Ulbrich, V. Subr, Adv Drug Deliv Rev 2010, 62, 150.

J. Kopecek, P. Kopecková, Adv Drug Deliv Rev 2010, 62, 122.

P. Chytil, L. Kostka, T. Etrych, J Pers Med 2021, 11, 115.

O. Lidický, P. Klener, D. Machová, P. Vocková, E. Pokorná, K. Helman, C. Mavis, O. Janousková, T. Etrych, J. Controlled Release 2020, 328, 160.

T. Etrych, J. Strohalm, L. Kovar, M. Kabesová, B. Rihova, K. Ulbrich, J. Controlled Release 2009, 140, 18.

M. Kovar, J. Strohalm, T. Etrych, K. Ulbrich, B. Rihova, Bioconjug Chem 2002, 13, 206.

L. Zhang, Y. Fang, J. Kopecek, J. Yang, Eur. J. Pharm. Sci. 2017, 103, 36.

K. Tappertzhofen, V. V. Metz, M. Hubo, M. Barz, R. Postina, H. Jonuleit, R. Zentel, Macromol. Biosci. 2013, 13, 203.

K. Ulbrich, J. Strohalm, V. Subr, D. Plocova, R. Duncan, B. Rihova, Macromol. Symp. 1996, 103, 177.

P. Chytil, S. Hoffmann, L. Schindler, L. Kostka, K. Ulbrich, H. Caysa, T. Mueller, K. Mäder, T. Etrych, J. Controlled Release 2013, 172, 504.

R. Pola, E. Böhmová, M. Filipová, M. Pechar, J. Pankrác, D. Vetvicka, T. Olejár, M. Kabesová, P. Poucková, L. Sefc, M. Zábrodský, O. Janousková, J. Boucek, T. Etrych, Pharmaceutics 2020, 12, 31.

P. Sacha, T. Knedlík, J. Schimer, J. Tykvart, J. Parolek, V. Navrátil, P. Dvorakova, F. Sedlák, K. Ulbrich, J. Strohalm, P. Majer, V. Subr, J. Konvalinka, Angew. Chem., Int. Ed. Engl. 2016, 55, 2356.

P. Chytil, T. Etrych, J. Kriz, V. Subr, K. Ulbrich, Eur. J. Pharm. Sci. 2010, 41, 473.

V. Subr, K. Ulbrich, React. Funct. Polym. 2006, 66, 1525.

V. Subr, T. Ormsby, P. Sacha, J. Konvalinka, T. Etrych, L. Kostka, Polym. Chem. 2021, 12, 6009.

G. L. Ellman, Arch. Biochem. Biophys. 1959, 82, 70.

T. Kalina, J. Flores-Montero, Q. Lecrevisse, C. E. Pedreira, V. H. J. Van Der Velden, M. Novakova, E. Mejstrikova, O. Hrusak, S. Böttcher, D. Karsch, L. Sedek, A. Trinquand, N. Boeckx, J. Caetano, V. Asnafi, P. Lucio, M. Lima, A. Helena Santos, P. Bonaccorso, A. J. Van Der Sluijs-Gelling, A. W. Langerak, M. Martin-Ayuso, T. Szczepanski, J. J. M. Van Dongen, A. Orfao, Cytometry, Part A 2015, 87, 145.

A. Postma, T. P. Davis, G. Moad, M. S. O'shea, Macromolecules 2005, 38, 5371.

Y. K. Chong, G. Moad, E. Rizzardo, S. H. Thang, Macromolecules 2007, 40, 4446.

E. Randárová, J. Kudlacova, T. Etrych, J. Controlled Release 2020, 325, 304.

M. Abdollahpour-Alitappeh, M. Lotfinia, S. Razavi-Vakhshourpour, S. Jahandideh, H. Najminejad, K. Sineh Sepehr, R. Moazami, E. Shams, M. Habibi-Anbouhi, M. Abolhassani, Iran Biomed. J. 2017, 21, 270.

M. M. C. Sun, K. S. Beam, C. G. Cerveny, K. J. Hamblett, R. S. Blackmore, M. Y. Torgov, F. G. M. Handley, N. C. Ihle, P. D. Senter, S. C. Alley, Bioconjugate Chem. 2005, 16, 1282.

J. E. Berlier, A. Rothe, G. Buller, J. Bradford, D. R. Gray, B. J. Filanoski, W. G. Telford, S. Yue, J. Liu, C.-Y. Cheung, W. Chang, J. D. Hirsch, J. M. Beechem, R. P. Haugland, R. P. Haugland, J. Histochem. Cytochem. 2003, 51, 1699.

T. Kalina, K. Fišer, M. Pérez-Andrés, D. Kuzílková, M. Cuenca, S. J. W. Bartol, E. Blanco, P. Engel, M. C. van Zelm, Front Immunol. 2019, 10, 2434.

D. Kuzilková, C. Bugarin, K. Rejlova, A. R. Schulz, H. E. Mei, M. Paganin, A. Biffi, A. Biondi, T. Kalina, G. Gaipa, Haematologica 2021, 107, 1293.

L. P. Arnett, R. Rana, W. W-Y. Chung, X. Li, M. Abtahi, D. Majonis, J. Bassan, M. Nitz, M. A. Winnik, Chem. Rev. 2023, 123, 1166.

M. Nováková, H. Glier, N. Brdicková, M. Vlková, A. H. Santos, M. Lima, M. Roussel, J. Flores-Montero, T. Szczepanski, S. Böttcher, V. H. J. Van Der Velden, P. Fernandez, E. Mejstříková, L. Burgos, B. Paiva, J. J. M. Van Dongen, A. Orfao, T. Kalina, J. Immunol. Methods 2019, 475, 112388.

K. E. Hally, L. Ferrer-Font, K. R. Pilkington, P. D. Larsen, Cytometry A 2022, 101, 374.

Tviblindi algorithm identifies branching developmental trajectories of human B-cell development and describes abnormalities in RAG-1 and WAS patients