Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development

Authors

DOI:

https://doi.org/10.62482/pmj.5

Keywords:

Flow cytometry, drug discovery, drug development , mass cytometry , spectral cytometry

Abstract

Flow cytometry, a pivotal tool in clinical and research labs since the discovery of cell markers in the mid-1970s, plays a crucial role across all phases of drug discovery. Modern flow cytometers can detect rare cell types relevant to disease pathogenesis, measure numerous parameters simultaneously, thus, offer versatility in drug screening. In drug discovery studies, flow cytometry contributes to the assessment of drug pharmacokinetics, pharmacodynamics and safety in animal models and clinical trials. It can also be used to monitor drug efficacy and identify biomarkers for diagnosis and prognosis.

In essence, flow cytometry is a versatile, instrumental technique that supports drug discovery from target identification through to clinical development, limited only by the creativity of the researcher and the availability of fluorescent labels or specific size/scatter related findings. This review article focuses on the use of flow cytometry in drug discovery and drug development studies, summarizing not only conventional assays such as immunophenotyping, measurement of programmed cell death pathways and cell division to provide insights into drug effects and patient responses, but also novel approaches including mass cytometry, spectral cytometry, and droplet cytometry.

Keywords: Flow cytometry, drug discovery, drug development, mass cytometry, spectral cytometry

References

Seyhan AA. Lost in translation: the valley of death across preclinical and clinical divide – identification of problems and overcoming obstacles. Transl Med Commun [Internet]. 2019 Dec 18;4(1):18. doi: 10.1186/s41231-019-0050-7

Tuschl H, Schwab CE. Flow cytometric methods used as screening tests for basal toxicity of chemicals. Toxicol Vitr [Internet]. 2004 Aug;18(4):483–91. doi: 10.1016/j.tiv.2003.12.004

Barnard RM. Flow cytometry: a flexible tool for biomarker research. Bioanalysis [Internet]. 2012 Oct;4(20):2471–83. doi: 10.4155/bio.12.225

Hilt E, Sun YS, McCloskey TW, Eck S, McIntosh T, Grugan KD, et al. Best practices for optimization and validation of flow cytometry‐based receptor occupancy assays. Cytom Part B Clin Cytom [Internet]. 2021 Jan;100(1):63–71. doi: 10.1002/cyto.b.21970

McCausland M, Lin Y-D, Nevers T, Groves C, Decman V. With great power comes great responsibility: high-dimensional spectral flow cytometry to support clinical trials. Bioanalysis [Internet]. 2021 Nov;13(21):1597–616. doi: 10.1002/cyto.b.21970

Antal-Szalmás P, Nagy B, Debreceni IB, Kappelmayer J. Measurement of Soluble Biomarkers by Flow Cytometry. EJIFCC [Internet]. 2013 Jan;23(4):135–42.

McKinnon KM. Flow Cytometry: An Overview. Curr Protoc Immunol [Internet]. 2018 Jan 21;120(1). doi: 10.1002/cpim.40

Streitz M, Miloud T, Kapinsky M, Reed MR, Magari R, Geissler EK, et al. Standardization of whole blood immune phenotype monitoring for clinical trials: panels and methods from the ONE study. Transplant Res [Internet]. 2013 Dec 25;2(1):17. doi: 10.1186/2047-1440-2-17

Lambert C, Yanikkaya Demirel G, Keller T, Preijers F, Psarra K, Schiemann M, et al. Flow Cytometric Analyses of Lymphocyte Markers in Immune Oncology: A Comprehensive Guidance for Validation Practice According to Laws and Standards. Front Immunol [Internet]. 2020 Sep 17;11. doi: 10.3389/fimmu.2020.02169

Rowley T. Flow Cytometry - A Survey and the Basics [Internet]. Vol. 2, Materials and Methods. 2012. doi: 10.13070/mm.en.2.125

Adan A, Alizada G, Kiraz Y, Baran Y, Nalbant A. Flow cytometry: basic principles and applications. Crit Rev Biotechnol [Internet]. 2017 Feb 17;37(2):163–76. doi: 10.3109/07388551.2015

Maecker HT, McCoy JP, Nussenblatt R. Standardizing immunophenotyping for the Human Immunology Project. Nat Rev Immunol [Internet]. 2012 Mar 17;12(3):191–200. doi: 10.1038/nri3158

Cossarizza A, Chang H, Radbruch A, Acs A, Adam D, Adam‐Klages S, et al. Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). Eur J Immunol [Internet]. 2019 Oct 21;49(10):1457–973. doi: 10.1002/eji.201970107

Newton HS, Dobrovolskaia MA. Immunophenotyping: Analytical approaches and role in preclinical development of nanomedicines. Adv Drug Deliv Rev [Internet]. 2022 Jun;185:114281. doi: 10.1016/j.addr.2022.114281

Iyer A, Hamers AAJ, Pillai AB. CyTOF® for the Masses. Front Immunol [Internet]. 2022 Apr 14;13. doi: 10.3389/fimmu.2022.815828

Hartmann FJ, Bendall SC. Immune monitoring using mass cytometry and related high-dimensional imaging approaches. Nat Rev Rheumatol [Internet]. 2020 Feb;16(2):87–99. doi: 10.1038/s41584-019-0338-z

Preglej T, Brinkmann M, Steiner G, Aletaha D, Göschl L, Bonelli M. Advanced immunophenotyping: A powerful tool for immune profiling, drug screening, and a personalized treatment approach. Front Immunol [Internet]. 2023 Mar 24;14. doi: 10.3389/fimmu.2023.1096096

Nagaoka K, Shirai M, Taniguchi K, Hosoi A, Sun C, Kobayashi Y, et al. Deep immunophenotyping at the single-cell level identifies a combination of anti-IL-17 and checkpoint blockade as an effective treatment in a preclinical model of data-guided personalized immunotherapy. J Immunother Cancer [Internet]. 2020 Oct 22;8(2):e001358. doi: 10.1136/jitc-2020-001358

Huang YV, Waliany S, Lee D, Galdos FX, Witteles RM, Neal JW, et al. The Role of Single-Cell Profiling and Deep Immunophenotyping in Understanding Immune Therapy Cardiotoxicity. JACC CardioOncology [Internet]. 2022 Dec;4(5):629–34. doi: 10.1016/j.jaccao.2022.08.012

Villani A-C, Satija R, Reynolds G, Sarkizova S, Shekhar K, Fletcher J, et al. Single-cell RNA-seq reveals new types of human blood dendritic cells, monocytes, and progenitors. Science (80- ) [Internet]. 2017 Apr 21;356(6335). doi: 10.1126/science.aah4573

Darzynkiewicz Z. Critical Aspects in Analysis of Cellular DNA Content. Curr Protoc Cytom [Internet]. 2011 Apr;56(1). doi: 10.1002/0471142956.cy0702s56

Camplejohn RS. Flow Cytometric Measurement of Cell Proliferation. In: Metastasis Research Protocols [Internet]. New Jersey: Humana Press; 2001. p. 133–43. doi: 10.1385/1-59259-136-1:133

Matson JP, Cook JG. Cell cycle proliferation decisions: the impact of single cell analyses. FEBS J [Internet]. 2017 Feb 5;284(3):362–75. doi: 10.1111/febs.13898

Telford W, Tamul K, Bradford J. Measurement and Characterization of Apoptosis by Flow Cytometry. Curr Protoc Cytom [Internet]. 2016 Jul;77(1). doi: 10.1002/cpcy.1

Telford WG, Komoriya A, Packard BZ. Multiparametric Analysis of Apoptosis by Flow and Image Cytometry. In: Flow Cytometry Protocols [Internet]. New Jersey: Humana Press; 2004. p. 141–60. doi: 10.1385/1-59259-773-4:141

Tario JD, Muirhead KA, Pan D, Munson ME, Wallace PK. Tracking Immune Cell Proliferation and Cytotoxic Potential Using Flow Cytometry. In 2011. p. 119–64. doi: 10.1007/978-1-61737-950-5_7

Solius GM, Maltsev DI, Belousov V V., Podgorny O V. Recent advances in nucleotide analogue-based techniques for tracking dividing stem cells: An overview. J Biol Chem [Internet]. 2021 Nov;297(5):101345. doi: 10.1016/j.jbc.2021.101345

Flomerfelt FA, Gress RE. Analysis of Cell Proliferation and Homeostasis Using EdU Labeling. In 2016. p. 211–20. doi: 10.1007/978-1-4939-2809-5_18

Lyons AB. Divided we stand: Tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester. Immunol Cell Biol [Internet]. 1999 Dec;77(6):509–15. doi: 10.1046/j.1440-1711.1999.00864.x

Quah BJC, Warren HS, Parish CR. Monitoring lymphocyte proliferation in vitro and in vivo with the intracellular fluorescent dye carboxyfluorescein diacetate succinimidyl ester. Nat Protoc [Internet]. 2007 Sep 23;2(9):2049–56. doi: 10.1038/nprot.2007.296

Sasaki K, Kurose A, Ishida Y. Flow cytometric analysis of the expression of PCNA during the cell cycle in hela cells and effects of the inhibition of DNA synthesis on it. Cytometry [Internet]. 1993 Nov 21;14(8):876–82. doi: 10.1002/cyto.990140805

Kim KH, Sederstrom JM. Assaying Cell Cycle Status Using Flow Cytometry. Curr Protoc Mol Biol [Internet]. 2015 Jul;111(1). doi: 10.1002/0471142727.mb2806s111

Wlodkowic D, Telford W, Skommer J, Darzynkiewicz Z. Apoptosis and Beyond: Cytometry in Studies of Programmed Cell Death. In 2011. p. 55–98. doi: 10.1016/B978-0-12-385493-3.00004-8

Kumar R, Saneja A, Panda AK. An Annexin V-FITC—Propidium Iodide-Based Method for Detecting Apoptosis in a Non-Small Cell Lung Cancer Cell Line. In 2021. p. 213–23. Available from: http://link.springer.com/10.1007/978-1-0716-1278-1_17

Wilkins RC, Kutzner BC, Truong M, Sanchez‐Dardon J, McLean JRN. Analysis of radiation‐induced apoptosis in human lymphocytes: Flow cytometry using Annexin V and propidium iodide versus the neutral comet assay. Cytometry [Internet]. 2002 May 17;48(1):14–9. Available from: https://onlinelibrary.wiley.com/doi/10.1002/cyto.10098

Rieger AM, Nelson KL, Konowalchuk JD, Barreda DR. Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death. J Vis Exp [Internet]. 2011 Apr 24;(50). doi: 10.3791/2597

Crowley LC, Marfell BJ, Scott AP, Waterhouse NJ. Quantitation of Apoptosis and Necrosis by Annexin V Binding, Propidium Iodide Uptake, and Flow Cytometry. Cold Spring Harb Protoc [Internet]. 2016 Nov;2016(11):pdb.prot087288. doi: 10.1101/pdb.prot087288

Lakshmanan I, Batra S. Protocol for Apoptosis Assay by Flow Cytometry Using Annexin V Staining Method. BIO-PROTOCOL [Internet]. 2013;3(6). doi: 10.21769/bioprotoc.374

Trahtemberg U, Atallah M, Krispin A, Verbovetski I, Mevorach D. Calcium, leukocyte cell death and the use of annexin V: fatal encounters. Apoptosis [Internet]. 2007 Oct 20;12(10):1769–80. doi: 10.1007/s10495-007-0097-1

Darzynkiewicz Z, Galkowski D, Zhao H. Analysis of apoptosis by cytometry using TUNEL assay. Methods [Internet]. 2008 Mar;44(3):250–4. doi: 10.1016/j.ymeth.2007.11.008

Mirzayans R, Murray D. Do TUNEL and Other Apoptosis Assays Detect Cell Death in Preclinical Studies? Int J Mol Sci [Internet]. 2020 Nov 29;21(23):9090. doi: 10.3390/ijms21239090

Kunzmann A, Liu D, Annett K, Malaisé M, Thaa B, Hyland P, et al. Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes. Immun Ageing [Internet]. 2006 Dec 19;3(1):8. doi: 10.1186/1742-4933-3-8

Belloc F, Belaud-Rotureau MA, Lavignolle V, Bascans E, Braz-Pereira E, Durrieu F, et al. Flow cytometry detection of caspase 3 activation in preapoptotic leukemic cells. Cytometry [Internet]. 2000 Jun 1;40(2):151–60. doi: 10.1002/(sici)1097-0320(20000601)40:2<151::aid-cyto9>3.0.co;2-9

Komoriya A, Packard BZ, Brown MJ, Wu M-L, Henkart PA. Assessment of Caspase Activities in Intact Apoptotic Thymocytes Using Cell-Permeable Fluorogenic Caspase Substrates. J Exp Med [Internet]. 2000 Jun 5;191(11):1819–28. doi: 10.1084/jem.191.11.1819

Amstad PA, Yu G, Johnson GL, Lee BW, Dhawan S, Phelps DJ. Detection of Caspase Activation In Situ by Fluorochrome-Labeled Caspase Inhibitors. Biotechniques [Internet]. 2001 Sep;31(3):608–16. doi: 10.2144/01313pf01

Bu F, Zhang J, Shuai W, Liu J, Sun Q, Ouyang L. Repurposing drugs in autophagy for the treatment of cancer: From bench to bedside. Drug Discov Today [Internet]. 2022 Jul;27(7):1815–31. Available from: https://linkinghub.elsevier.com/retrieve/pii/S1359644621004943

Kocak M, Ezazi Erdi S, Jorba G, Maestro I, Farrés J, Kirkin V, et al. Targeting autophagy in disease: established and new strategies. Autophagy [Internet]. 2022 Mar 4;18(3):473–95. doi: 10.1016/j.drudis.2021.11.013

Kaminskyy VO. A Quantitative Flow Cytometry–Based Method for Autophagy Detection Across the Cell Cycle. In: Methods in Molecular Biology [Internet]. Clifton, NJ; 2022. p. 65–74. doi: 10.1007/978-1-0716-2071-7_5

Koepke L, Winter B, Grenzner A, Regensburger K, Engelhart S, van der Merwe JA, et al. An improved method for high-throughput quantification of autophagy in mammalian cells. Sci Rep [Internet]. 2020 Jul 22;10(1):12241. doi: 10.1038/s41598-020-68607-w

Demishtein A, Porat Z, Elazar Z, Shvets E. Applications of flow cytometry for measurement of autophagy. Methods [Internet]. 2015 Mar;75:87–95. doi: 10.1016/j.ymeth.2014.12.020

Xu F, Fang Y, Yan L, Xu L, Zhang S, Cao Y, et al. Nuclear localization of Beclin 1 promotes radiation-induced DNA damage repair independent of autophagy. Sci Rep [Internet]. 2017 Mar 27;7(1):45385. https://doi.org/10.1038/srep45385

He Y, Mo Z, Xue Z, Fang Y. Establish a flow cytometric method for quantitative detection of Beclin-1 expression. Cytotechnology [Internet]. 2013 Aug 23;65(4):481–9. doi: 10.1007/s10616-012-9503-9

Mizushima N, Murphy LO. Autophagy Assays for Biological Discovery and Therapeutic Development. Trends Biochem Sci [Internet]. 2020 Dec;45(12):1080–93. doi: 10.1016/j.tibs.2020.07.006

Engedal N, Sønstevold T, Beese CJ, Selladurai S, Melcher T, Simensen JE, et al. Measuring Autophagic Cargo Flux with Keima-Based Probes. In 2022. p. 99–115. doi: 10.1007/978-1-0716-2071-7_7

Gu Y, Li Y, Wang J, Zhang L, Zhang J, Wang Y. Targeting ferroptosis: Paving new roads for drug design and discovery. Eur J Med Chem [Internet]. 2023 Feb;247:115015. doi: 10.1016/j.ejmech.2022.115015

Li J, Cao F, Yin H, Huang Z, Lin Z, Mao N, et al. Ferroptosis: past, present and future. Cell Death Dis [Internet]. 2020 Feb 3;11(2):88. doi: 10.1038/s41419-020-2298-2

Dixon SJ, Stockwell BR. The Hallmarks of Ferroptosis. Annu Rev Cancer Biol [Internet]. 2019 Mar 4;3(1):35–54. https://doi.org/10.1146/annurev-cancerbio-030518-055844

Hadian K, Stockwell BR. A roadmap to creating ferroptosis-based medicines. Nat Chem Biol [Internet]. 2021 Nov 21;17(11):1113–6. doi: 10.1038/s41589-021-00853-z

Dai Z, Zhang W, Zhou L, Huang J. Probing Lipid Peroxidation in Ferroptosis: Emphasizing the Utilization of C11-BODIPY-Based Protocols. In: Methods in Molecular Biology [Internet]. 2023. p. 61–72. doi: 10.1007/978-1-0716-3433-2_6

Martinez AM, Kim A, Yang WS. Detection of Ferroptosis by BODIPYTM 581/591 C11. In: Methods in Molecular Biology [Internet]. 2020. p. 125–30. doi: 10.1007/978-1-0716-0247-8_11

Yamanaka K, Saito Y, Sakiyama J, Ohuchi Y, Oseto F, Noguchi N. A novel fluorescent probe with high sensitivity and selective detection of lipid hydroperoxides in cells. RSC Adv [Internet]. 2012;2(20):7894. DOI=c2ra20816d

Chen X, Comish PB, Tang D, Kang R. Characteristics and Biomarkers of Ferroptosis. Front Cell Dev Biol [Internet]. 2021 Jan 21;9. doi: 10.3389/fcell.2021.637162

Tan Y, Chen Q, Li X, Zeng Z, Xiong W, Li G, et al. Pyroptosis: a new paradigm of cell death for fighting against cancer. J Exp Clin Cancer Res [Internet]. 2021 May 3;40(1):153. https://doi.org/10.1186/s13046-021-01959-x

Chan AH, Schroder K. Inflammasome signaling and regulation of interleukin-1 family cytokines. J Exp Med [Internet]. 2020 Jan 6;217(1). doi: 10.1084/jem.20190314

He W, Wan H, Hu L, Chen P, Wang X, Huang Z, et al. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res [Internet]. 2015 Dec 27;25(12):1285–98. https://doi.org/10.1038/cr.2015.139

Malhotra S, Hurtado-Navarro L, Pappolla A, Villar LMM, Río J, Montalban X, et al. Increased NLRP3 Inflammasome Activation and Pyroptosis in Patients With Multiple Sclerosis With Fingolimod Treatment Failure. Neurol Neuroimmunol Neuroinflammation [Internet]. 2023 May;10(3). doi: 10.1212/NXI.0000000000200100

Van Schoor E, Ospitalieri S, Moonen S, Tomé SO, Ronisz A, Ok O, et al. Increased pyroptosis activation in white matter microglia is associated with neuronal loss in ALS motor cortex. Acta Neuropathol [Internet]. 2022 Sep 22;144(3):393–411. doi: 10.1007/s00401-022-02466-9

Zhuang J, Cui H, Zhuang L, Zhai Z, Yang F, Luo G, et al. Bronchial epithelial pyroptosis promotes airway inflammation in a murine model of toluene diisocyanate-induced asthma. Biomed Pharmacother [Internet]. 2020 May;125:109925. doi: 10.1016/j.biopha.2020.109925

Lu L, Zhang Y, Tan X, Merkher Y, Leonov S, Zhu L, et al. Emerging mechanisms of pyroptosis and its therapeutic strategy in cancer. Cell Death Discov [Internet]. 2022 Jul 27;8(1):338. doi: 10.1038/s41420-022-01101-6

Wei S, Feng M, Zhang S. Molecular Characteristics of Cell Pyroptosis and Its Inhibitors: A Review of Activation, Regulation, and Inhibitors. Int J Mol Sci [Internet]. 2022 Dec 17;23(24):16115. doi: 10.3390/ijms232416115

Li T, Zheng G, Li B, Tang L. Pyroptosis: A promising therapeutic target for noninfectious diseases. Cell Prolif [Internet]. 2021 Nov 29;54(11). doi: 10.1111/cpr.13137

Rosli S, Harpur CM, Lam M, West AC, Hodges C, Mansell A, et al. Gasdermin D promotes hyperinflammation and immunopathology during severe influenza A virus infection. Cell Death Dis [Internet]. 2023 Nov 9;14(11):727. doi: 10.1038/s41419-023-06258-1

Keitelman IA, Shiromizu CM, Zgajnar NR, Danielián S, Jancic CC, Martí MA, et al. The interplay between serine proteases and caspase-1 regulates the autophagy-mediated secretion of Interleukin-1 beta in human neutrophils. Front Immunol [Internet]. 2022 Aug 25;13. doi: 10.3389/fimmu.2022.832306

Liu TJ, Lin LL, McMeniman E, Wu J, Kao Y-C, Kumari S, et al. Cytokine/Chemokine assessment as a complementary diagnostic tool for inflammatory skin diseases. Front Immunol [Internet]. 2022 Nov 16;13. doi: 10.3389/fimmu.2022.1028435

Usher PA, Galsgaard ED, Kruse K, Wang J, Krogh BO, Mandelbaum J, et al. Sensitive and Specific In Situ Hybridization for Early Drug Discovery. In 2014. p. 103–23. doi: 10.1007/978-1-4939-1459-3_10

Baerlocher GM, Vulto I, de Jong G, Lansdorp PM. Flow cytometry and FISH to measure the average length of telomeres (flow FISH). Nat Protoc [Internet]. 2006 Dec 21;1(5):2365–76. doi: 10.1038/nprot.2006.263

Rufer N, Dragowska W, Thornbury G, Roosnek E, Lansdorp PM. Telomere length dynamics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotechnol [Internet]. 1998 Aug;16(8):743–7. doi: 10.1038/nbt0898-743

Pereira AC, Tenreiro A, Cunha M V. When FLOW-FISH met FACS: Combining multiparametric, dynamic approaches for microbial single-cell research in the total environment. Sci Total Environ [Internet]. 2022 Feb;806:150682. doi: 10.1016/j.scitotenv.2021.150682

Arrigucci R, Bushkin Y, Radford F, Lakehal K, Vir P, Pine R, et al. FISH-Flow, a protocol for the concurrent detection of mRNA and protein in single cells using fluorescence in situ hybridization and flow cytometry. Nat Protoc [Internet]. 2017 Jun 18;12(6):1245–60. doi: 10.1038/nprot.2017.039

Freen-van Heeren JJ. Flow-FISH as a Tool for Studying Bacteria, Fungi and Viruses. BioTech [Internet]. 2021 Oct 11;10(4):21. https://doi.org/10.3390/biotech10040021

Gutierrez-Rodrigues F, Santana-Lemos BA, Scheucher PS, Alves-Paiva RM, Calado RT. Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans. Lustig AJ, editor. PLoS One [Internet]. 2014 Nov 19;9(11):e113747. doi: 10.1371/journal.pone.0113747

Ferreira MSV, Kirschner M, Halfmeyer I, Estrada N, Xicoy B, Isfort S, et al. Comparison of flow‐FISH and MM–qPCR telomere length assessment techniques for the screening of telomeropathies. Ann N Y Acad Sci [Internet]. 2020 Apr 24;1466(1):93–103. doi: 10.1111/nyas.14248

CEDEÑO-ARIAS M. Validation of a Flow Cytometry Based Binding Assay for Evaluation of Monoclonal Antibody Recognizing EGF Receptor. Sci Pharm [Internet]. 2011;79(3):569–81. https://doi.org/10.3797/scipharm.1104-18

Sprokholt JK, Hertoghs N, Geijtenbeek TBH. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity. In: Methods in Molecular Biology [Internet]. 2016. p. 121–9. Available from: http://link.springer.com/10.1007/978-1-4939-3335-8_8

Mire-Sluis AR. Progress in the use of biological assays during the development of biotechnology products. Pharm Res [Internet]. 2001 Sep;18(9):1239–46. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11683235

Stewart JJ, Green CL, Jones N, Liang M, Xu Y, Wilkins DEC, et al. Role of receptor occupancy assays by flow cytometry in drug development. Cytom Part B Clin Cytom [Internet]. 2016 Mar 16;90(2):110–6. Available from: https://onlinelibrary.wiley.com/doi/10.1002/cyto.b.21355

Moulard M, Ozoux M. How validated receptor occupancy flow cytometry assays can impact decisions and support drug development. Cytom Part B Clin Cytom [Internet]. 2016 Mar 11;90(2):150–8. doi: 10.1007/978-1-4939-3335-8_8

Liang M, Schwickart M, Schneider AK, Vainshtein I, Del Nagro C, Standifer N, et al. Receptor occupancy assessment by flow cytometry as a pharmacodynamic biomarker in biopharmaceutical development. Cytom Part B Clin Cytom [Internet]. 2016 Mar 31;90(2):117–27. doi: 10.1002/cyto.b.21259

Zhao Z, Fu J, Dhakal S, Johnson-Buck A, Liu M, Zhang T, et al. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion. Nat Commun [Internet]. 2016 Feb 10;7(1):10619. doi: 10.1038/ncomms10619

Gorry M, Yoneyama T, Vujanovic L, Moss ML, Garlin MA, Miller MA, et al. Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells. J Cancer [Internet]. 2020;11(3):702–15. doi: 10.7150/jca.30813

Telford W, Cox W, Singer V. Detection of endogenous and antibody-conjugated alkaline phosphatase with ELF-97 phosphate in multicolor flow cytometry applications. Cytometry [Internet]. 2001 Feb 1;43(2):117–25. Available from: http://www.ncbi.nlm.nih.gov/pubmed/11169576

Farinas E. Fluorescence Activated Cell Sorting for Enzymatic Activity. Comb Chem High Throughput Screen [Internet]. 2006 May 1;9(4):321–8.

Kovarik ML, Allbritton NL. Measuring enzyme activity in single cells. Trends Biotechnol [Internet]. 2011 May;29(5):222–30. Available from: https://linkinghub.elsevier.com/retrieve/pii/S0167779911000138

Grupillo M, Lakomy R, Geng X, Styche A, Rudert WA, Trucco M, et al. An improved intracellular staining protocol for efficient detection of nuclear proteins in YFP-expressing cells. Biotechniques [Internet]. 2011 Dec;51(6):417–20. doi: 10.2174/138620706776843200

Renner P, Crone M, Kornas M, Pioli KT, Pioli PD. Intracellular flow cytometry staining of antibody-secreting cells using phycoerythrin-conjugated antibodies: pitfalls and solutions. Antib Ther [Internet]. 2022 Aug 1;5(3):151–63. doi: 10.1093/abt/tbac013

L. Chang R, Yeh C-H, Albitar M. Quantification of Intracellular Proteins and Monitoring Therapy Using Flow Cytometry. Curr Drug Targets [Internet]. 2010 Aug 1;11(8):994–9. doi: 10.2174/138945010791591296

Lamoreaux L, Roederer M, Koup R. Intracellular cytokine optimization and standard operating procedure. Nat Protoc [Internet]. 2006 Aug 9;1(3):1507–16. doi: 10.1038/nprot.2006.268

Yin Y, Mitson‐Salazar A, Prussin C. Detection of Intracellular Cytokines by Flow Cytometry. Curr Protoc Immunol [Internet]. 2015 Aug 3;110(1). doi:10.1002/0471142735.im0624s110

Hedley BD, Keeney M. Technical issues: flow cytometry and rare event analysis. Int J Lab Hematol [Internet]. 2013 Jun 17;35(3):344–50. doi: 10.1111/ijlh.12068

Francis C, Connelly MC. Rapid single-step method for flow cytometric detection of surface and intracellular antigens using whole blood. Cytometry [Internet]. 1996 Sep 1;25(1):58–70. doi: 10.1002/(SICI)1097-0320(19960901)25:1<58::AID-CYTO7>3.0.CO;2-A

Sedlmayr P, Grosshaupt B, Muntean W. Flow cytometric detection of intracellular platelet antigens. Cytometry [Internet]. 1996 Apr 1;23(4):284–9. doi: 10.1002/(SICI)1097-0320(19960401)23:4<284::AID-CYTO4>3.0.CO;2-H

Krug N, Thurau AM, Lackie P, Baier J, Schultze-Werninghaus G, Rieger CHL, et al. A flow cytometric method for the detection of intracellular basic proteins in unseparated peripheral blood and bone marrow eosinophils. J Immunol Methods [Internet]. 1996 Apr;190(2):245–54. doi: 10.1016/0022-1759(95)00272-3

Ruitenberg JJ, Waters CA. A rapid flow cytometric method for the detection of intracellular cyclooxygenases in human whole blood monocytes and a COX-2 inducible human cell line. J Immunol Methods [Internet]. 2003 Mar;274(1–2):93–104. doi: 10.1016/s0022-1759(02)00507-0

Lafarge S, Hamzeh-Cognasse H, Chavarin P, Genin C, Garraud O, Cognasse F. A flow cytometry technique to study intracellular signals NF-κB and STAT3 in peripheral blood mononuclear cells. BMC Mol Biol [Internet]. 2007;8(1):64. doi: 10.1186/1471-2199-8-64

Cavalcanti GB, Scheiner MAM, Simões Magluta EP, Vasconcelos F da C, Klumb CE, Maia RC. p53 flow cytometry evaluation in leukemias: Correlation to factors affecting clinical outcome. Cytom Part B Clin Cytom [Internet]. 2010 Jul 2;78B(4):253–9. doi: 10.1002/cyto.b.20514

Di Rosa F, Cossarizza A, Hayday AC. To Ki or Not to Ki: Re-Evaluating the Use and Potentials of Ki-67 for T Cell Analysis. Front Immunol [Internet]. 2021 Apr 9;12. doi: 10.3389/fimmu.2021.653974

Vanhulle E, Provinciael B, Stroobants J, Camps A, Maes P, Vermeire K. Intracellular flow cytometry complements RT-qPCR detection of circulating SARS-CoV-2 variants of concern. Biotechniques [Internet]. 2022 Jun;72(6):245–54. doi: 10.2144/btn-2022-0018

Wang W, Zhang C-X, Li Z-L, Gong M, Ma Y-G. Detection of intracellular IgD using flow cytometry could be a novel and supplementary method to diagnose IgD multiple myeloma. BMC Cancer [Internet]. 2018 Dec 11;18(1):650. doi: 10.1186/s12885-018-4562-8

Krutzik PO, Nolan GP. Intracellular phospho‐protein staining techniques for flow cytometry: Monitoring single cell signaling events. Cytom Part A [Internet]. 2003 Oct 17;55A(2):61–70. doi: 10.1002/cyto.a.10072

Krutzik PO, Irish JM, Nolan GP, Perez OD. Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol [Internet]. 2004 Mar;110(3):206–21. doi: 10.1016/j.clim.2003.11.009

Toney NJ, Schlom J, Donahue RN. Phosphoflow cytometry to assess cytokine signaling pathways in peripheral immune cells: potential for inferring immune cell function and treatment response in patients with solid tumors. J Exp Clin Cancer Res [Internet]. 2023 Sep 23;42(1):247. doi: 10.1186/s13046-023-02802-1

Wu S, Jin L, Vence L, Radvanyi LG. Development and application of ‘phosphoflow’ as a tool for immunomonitoring. Expert Rev Vaccines [Internet]. 2010 Jun 9;9(6):631–43. doi: 10.1586/erv.10.59

Fais S, O’Driscoll L, Borras FE, Buzas E, Camussi G, Cappello F, et al. Evidence-Based Clinical Use of Nanoscale Extracellular Vesicles in Nanomedicine. ACS Nano [Internet]. 2016 Apr 26;10(4):3886–99. doi: 10.1021/acsnano.5b08015

Yáñez‐Mó M, Siljander PR ‐M., Andreu Z, Bedina Zavec A, Borràs FE, Buzas EI, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles [Internet]. 2015 Jan 14;4(1). doi: 10.3402/jev.v4.27066

Tertel T, Görgens A, Giebel B. Analysis of individual extracellular vesicles by imaging flow cytometry. In: Methods in Enzymology [Internet]. 2020. p. 55–78. doi: 10.1016/bs.mie.2020.05.013

Morales-Kastresana A, Jones JC. Flow Cytometric Analysis of Extracellular Vesicles. In: Methods in Molecular Biology [Internet]. 2017. p. 215–25. doi: 10.1007/978-1-4939-6728-5_16

Lässer C, Eldh M, Lötvall J. Isolation and Characterization of RNA-Containing Exosomes. J Vis Exp [Internet]. 2012 Jan 9;(59). doi: 10.3791/3037

Ali Moussa HY, Manaph N, Ali G, Maacha S, Shin KC, Ltaief SM, et al. Single Extracellular Vesicle Analysis Using Flow Cytometry for Neurological Disorder Biomarkers. Front Integr Neurosci [Internet]. 2022 May 17;16. doi: 10.3389/fnint.2022.879832

Welsh JA, Van Der Pol E, Arkesteijn GJA, Bremer M, Brisson A, Coumans F, et al. MIFlowCyt‐EV: a framework for standardized reporting of extracellular vesicle flow cytometry experiments. J Extracell Vesicles [Internet]. 2020 Sep 3;9(1). doi: 10.1080/20013078.2020.1713526

Gustafsdottir SM, Ljosa V, Sokolnicki KL, Anthony Wilson J, Walpita D, Kemp MM, et al. Multiplex Cytological Profiling Assay to Measure Diverse Cellular States. Mancini MA, editor. PLoS One [Internet]. 2013 Dec 2;8(12):e80999. https://doi.org/10.1371/journal.pone.0080999

Willis C, Nyffeler J, Harrill J. Phenotypic Profiling of Reference Chemicals across Biologically Diverse Cell Types Using the Cell Painting Assay. SLAS Discov [Internet]. 2020 Aug;25(7):755–69. doi: 10.1177/2472555220928004

Bray M-A, Singh S, Han H, Davis CT, Borgeson B, Hartland C, et al. Cell Painting, a high-content image-based assay for morphological profiling using multiplexed fluorescent dyes. Nat Protoc [Internet]. 2016 Sep 25;11(9):1757–74. doi: 10.1038/nprot.2016.105

McMahon CL, Esqueda M, Yu J-J, Wall G, Romo JA, Vila T, et al. Development of an Imaging Flow Cytometry Method for Fungal Cytological Profiling and Its Potential Application in Antifungal Drug Development. J Fungi [Internet]. 2023 Jun 30;9(7):722. doi: 10.3390/jof9070722

Li L, Wang S, Xue J, Lin Y, Su L, Xue C, et al. Development of Spectral Nano-Flow Cytometry for High-Throughput Multiparameter Analysis of Individual Biological Nanoparticles. Anal Chem [Internet]. 2023 Feb 14;95(6):3423–33. doi: 10.1021/acs.analchem.2c05159

Hernández P, Gorrochategui J, Primo D, Robles A, Rojas JL, Espinosa AB, et al. Drug Discovery Testing Compounds in Patient Samples by Automated Flow Cytometry. SLAS Technol [Internet]. 2017 Jun;22(3):325–37. doi: 10.1177/2472630317700346

Young SM, Bologa C, Prossnitz ER, Oprea TI, Sklar LA, Edwards BS. High-Throughput Screening with HyperCyt® Flow Cytometry to Detect Small Molecule Formylpeptide Receptor Ligands. SLAS Discov [Internet]. 2005 Jun;10(4):374–82. doi: 10.1177/1087057105274532

Edwards BS, Sklar LA. Flow Cytometry: Impact on Early Drug Discovery. SLAS Discov [Internet]. 2015 Jul;20(6):689–707. doi: 10.1177/1087057115578273

van der Pan K, Khatri I, de Jager AL, Louis A, Kassem S, Naber BAE, et al. Performance of spectral flow cytometry and mass cytometry for the study of innate myeloid cell populations. Front Immunol [Internet]. 2023 May 19;14. doi: 10.3389/fimmu.2023.1191992

Di Zeo-Sánchez DE, Sánchez-Núñez P, Stephens C, Lucena MI. Characterizing Highly Cited Papers in Mass Cytometry through H-Classics. Biology (Basel) [Internet]. 2021 Feb 2;10(2):104. doi: 10.3390/biology10020104

Watson ECR, Baker W, Ahern D, Loi D, Cribbs AP, Oppermann U. Mass cytometry as a tool in target validation and drug discovery. In 2023. p. 541–74. doi: 10.1016/bs.mie.2023.07.006

Nolan JP. The evolution of spectral flow cytometry. Cytom Part A [Internet]. 2022 Oct 14;101(10):812–7. https://doi.org/10.1002/cyto.a.24566

Bonilla DL, Reinin G, Chua E. Full Spectrum Flow Cytometry as a Powerful Technology for Cancer Immunotherapy Research. Front Mol Biosci. 2021 Jan 29;7.

Li M, Liu H, Zhuang S, Goda K. Droplet flow cytometry for single-cell analysis. RSC Adv. 2021;11(34):20944–60. https://doi.org/10.3389/fmolb.2020.612801

Downloads

Published

2024-02-29

How to Cite

Aru, B., & Yanikkaya Demirel, G. (2024). Flow Cytometry: A Versatile and Powerful Tool for Drug Discovery and Development. Pharmedicine Journal, 1(1), 1–19. https://doi.org/10.62482/pmj.5

Issue

Section

Review