Antibody labeling kits vs conventional labeling: A comparative study in immunofluorescence of cell cultures

Kubilay Doğan Kılıç; Ayşegül Taşkıran Özyazıcı; Büşra Horuz

doi:10.19161/etd.1599016

EN TR

Antibody labeling kits vs conventional labeling: A comparative study in immunofluorescence of cell cultures

Abstract

Aim: This study aims to evaluate the comparative performance of antibody labeling kits and conventional antibody labeling methods regarding efficiency, accuracy, and practical usability in immunofluorescence applications on cell cultures. Materials and Methods: Both labeling methods were applied to identical cell culture samples. Labeling efficiency, fluorescence intensity, and antibody specificity were assessed using quantitative fluorescence microscopy. Practical aspects such as ease of use, time efficiency, and cost were also systematically evaluated. Results: Antibody labeling kits significantly reduced preparation time while increasing labeling efficiency, achieving comparable results to conventional methods in fluorescence intensity and antibody specificity. In sum, it is reported that it is higher ease of use for Antibody Labeling Kits. Conclusion: Antibody labeling kits offer a reliable and user-friendly alternative for immunofluorescence studies. By enhancing labeling efficiency and simplifying operational processes, these kits can accelerate research workflows and improve experimental outcomes in cell-based assays.

Keywords

Antikor etiketleme kitleri ile geleneksel etiketleme: Hücre kültürlerinde karşılaştırmalı immünofloresans bir çalışma

Öz

Amaç: Bu çalışma, antikor işaretleme kitleri ile geleneksel antikor işaretleme yöntemlerinin immün floresans uygulamalarında hücre kültürleri üzerindeki verimlilik, doğruluk ve pratik kullanılabilirlik açısından karşılaştırmalı performansını değerlendirmeyi amaçlamaktadır. Gereç ve Yöntem: Çalışmada her iki işaretleme yöntemi, aynı hücre kültürü örneklerine uygulanmıştır. İşaretleme verimliliği, floresans yoğunluğu ve antikor özgüllüğü, nicel floresans mikroskopisi ile değerlendirilmiştir. Ayrıca kullanım kolaylığı, zaman verimliliği ve maliyet gibi pratik unsurlar incelenmiştir. Bulgular: Antikor işaretleme kitleri, hazırlık süresini önemli ölçüde azaltırken işaretleme verimliliğini arttırmış ve floresans yoğunluğu ile antikor özgüllüğü açısından geleneksel yöntemlere eşdeğer sonuçlar sağlamıştır. Bütün değişkenler bir arada değerlendirildiğinde, antikor İşaretleme Kitlerinin kullanım kolaylığını daha yüksek olarak bildirmiştir. Sonuç: Antikor işaretleme kitleri, immün floresans çalışmaları için güvenilir ve kullanıcı dostu bir alternatif sunmaktadır. Etiketleme verimliliğini artırmaları ve operasyonel süreçleri basitleştirmeleri, bu kitlerin araştırma süreçlerini hızlandırarak hücre temelli deneylerde daha iyi sonuçlar elde edilmesini sağlayabileceğini göstermektedir.

Anahtar Kelimeler

References

Berg EA, Fishman JB. Labeling antibodies. Cold Spring Harb Protoc. 2020;2020(7):pdb-top099242.
Joshi S, Yu D. Immunofluorescence. In: Basic Science Methods for Clinical Researchers. Academic Press; 2017. p. 135-150.
Donaldson, J. G. (2015). Immunofluorescence staining. Current protocols in cell biology, 69(1), 4-270 3. FlexAble Antibody Labeling Kits Data Sheet, Available at: https://www.ptglab.com/products/flexable-antibody- labeling273 kits/?gclid=CjwKCAiAudG5BhAREiwAWMlSjJH9T9LnrZxuobVGuE2mIZGSOIWV1d- WjudcZAEA8cn785K4UBoq9BoCWn4QAvD_BwE Access Date: 20.09.2024
Alexa Fluor® 647 Conjugation Kit (Fast) - Lightning-Link® Data Sheet, Available at:
https://www.abcam.com/en-us/products/conjugation-kits/alexa-fluor-647-conjugation-kit-fast277 lightning-link-ab269823 Access Date: 20.09.2024
Yu S, Gao J, Wang H, Liu L, Liu X, Xu Y, et al. Significance of liver zonation in hepatocellular carcinoma. Front Cell Dev Biol. 2022;10:806408. doi:10.3389/fcell.2022.806408.
Watts LP, Natsume T, Saito Y, Garzon J, Dong Q, Boteva L, et al. The RIF1-long splice variant promotes G1 phase 53BP1 nuclear bodies to protect against replication stress. eLife.2020;9:e58020. doi:10.7554/eLife.58020.
Fan Y, Sun Z, Conrad F, Wen W, Zhao L, Lou J, et al. Multicolor fluorescence activated cell sorting to generate humanized monoclonal antibody binding seven subtypes of BoNT/F. PLoS One. 2022;17(9):e0273512.

Heo K, Min SW, Sung HJ, Kim HG, Kim HJ, Kim YH, et al. An aptamer-antibody complex (oligobody) as a novel delivery platform for targeted cancer therapies. J Control Release. 2016;229:1-9.
Bianchetti L, Marini MA, Isgrò M, Bellini A, Schmidt M, Mattoli S. IL-33 promotes the migration and proliferation of circulating fibrocytes from patients with allergen-exacerbated asthma. Biochem Biophys Res Commun. 2012;426(1):116-21.
Hemadi A, Ekrami A, Oormazdi H, Meamar AR, Akhlaghi L, Samarbaf-Zadeh AR, et al. Bioconjugated fluorescent silica nanoparticles for the rapid detection of Entamoeba histolytica. Acta Trop. 2015;145:26-30.
Guo Q, Li P, Chen M, et al. Exosomes from human umbilical cord stem cells suppress macrophage295 to-myofibroblast transition, alleviating renal fibrosis. Inflammation. 2024. doi:10.1007/s10753-024-02027-0.
Li Y, Jiang M, Wei Y, He X, Li G, Lu C, Ge D. Integrative analyses of pyrimidine salvage pathway298 related genes revealing the associations between UPP1 and tumor microenvironment. J Inflamm Res. 2024;17:101- 119. doi:10.2147/JIR.S440295.
Ichinose S, Susuki Y, Hosoi N, Kaneko R, Ebihara M, Hirai H, et al. Interaction between Teneurin-2 and microtubules via EB proteins provides a platform for GABAA receptor exocytosis. Elife. 2023 Jun 5;12.
Yurlova L, Metterlein M, Callahan D. 76 A novel method for antibody labeling 303 using Proteintech FlexAble kits. 2024 [cited 2025 Feb 25]; Available from: https://jitc.bmj.com/content/12/Suppl_2/A83.abstract
Kothurkar AA, Patient GS, Noel NCL, Krzywańskakrzywańska AM, Carr BJ, Chu CJ, et al. ’Iterative Bleaching Extends Multiplexity’facilitates simultaneous identification of all major retinal cell types. journals.biologists.comAA Kothurkar, GS Patient, NCL Noel, AM Krzywańska, BJ Carr, CJ Chu, RB MacDonaldJournal of Cell Science, 2024•journals.biologists.com [Internet]. 2024 [cited 2025 Feb 25]; Available from: https://journals.biologists.com/jcs/article/137/23/jcs263407/363401
Tobe-Nishimoto A, Morita Y, Nishimura J, Kitahira Y, Shun Takayama ·, Kishimoto · Satoko, et al. Tumor microenvironment dynamics in oral cancer: unveiling the role of inflammatory cytokines in a syngeneic mouse model. SpringerA Tobe-Nishimoto, Y Morita, J Nishimura, Y Kitahira, S Takayama, S KishimotoClinical & Experimental Metastasis, 2024•Springer [Internet]. 123AD Dec 1 [cited 2025 Feb 25];41:891–908. Available from: https://link.springer.com/article/10.1007/s10585- 024-10306-1
Sahoo, H. (2012). Fluorescent labeling techniques in biomolecules: a flashback. RSC advances, 2(18), 7017- 7029.
Wei W, Younis M, Lan X, ... JLJ of N, 2022 undefined. Single-domain antibody theranostics on the horizon. jnm.snmjournals.orgW Wei, MH Younis, X Lan, J Liu, W CaiJournal of Nuclear Medicine, 2022•jnm.snmjournals.org [Internet]. 2022 [cited 2025 Feb 25];63:1475–9. Available from: https://jnm.snmjournals.org/content/63/10/1475.abstract
Eng J, Bucher E, Hu Z, Zheng T, Gibbs SL, Chin K, et al. A framework for multiplex imaging optimization and reproducible analysis. nature.comJ Eng, E Bucher, Z Hu, T Zheng, SL Gibbs, K Chin, JW GrayCommunications biology, 2022•nature.com [Internet]. [cited 2025 Feb 25]; Available from: https://www.nature.com/articles/s42003-022-03368-y
Ramos-Vara J, pathology MMV, 2014 undefined. When tissue antigens and antibodies get along: revisiting the technical aspects of immunohistochemistry—the red, brown, and blue technique. journals.sagepub.comJA Ramos-Vara, MA MillerVeterinary pathology, 2014•journals.sagepub.com [Internet]. 2014 Jan [cited 2025 Feb 25];51(1):42–87. Available from: https://journals.sagepub.com/doi/abs/10.1177/0300985813505879
Meder F, Thomas SS, Fitzpatrick LW, Alahmari A, Wang S, Beirne JG, et al. Labeling the structural integrity of nanoparticles for advanced in situ tracking in bionanotechnology. ACS PublicationsF Meder, SS Thomas, LW Fitzpatrick, A Alahmari, S Wang, JG Beirne, G Vaz, G RedmondACS nano, 2016•ACS Publications [Internet]. 2016 Apr 26 [cited 2025 Feb 25];10(4):4660–71. Available from: https://pubs.acs.org/doi/abs/10.1021/acsnano.6b01001
Meuter S, Eberl M, of BMP of the NA, 2010 undefined. Prolonged antigen survival and cytosolic export in cross-presenting human γδ T cells. pnas.orgS Meuter, M Eberl, B MoserProceedings of the National Academy of Sciences, 2010•pnas.org [Internet]. 2010 May 11 [cited 2025 Feb 25];107(19):8730–5. Available from: https://www.pnas.org/doi/abs/10.1073/pnas.1002769107
Rivest F, Eroglu D, Pelz B, Kowal J, Kehren A, Navikas V, et al. Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. nature.comF Rivest, D Eroglu, B Pelz, J Kowal, A Kehren, V Navikas, MG Procopio, P BordignonScientific Reports, 2023•nature.com [Internet]. 123AD Dec 1 [cited 2025 Feb 25];13(1):16994. Available from: https://www.nature.com/articles/s41598-023- 43435-w
Liu S, Hoess P, Ries J. Super-Resolution Microscopy for Structural Cell Biology. Annu Rev Biophys. 2022;51:301–26.
Thorley, J. A., Pike, J., & Rappoport, J. Z. (2014). Super-resolution microscopy: a comparison of commercially available options. In Fluorescence microscopy (pp. 199-212). Academic Press
Werner C, Sauer M, Reviews CGC, 2021 undefined. Super-resolving microscopy in neuroscience. ACS PublicationsC Werner, M Sauer, C GeisChemical Reviews, 2021•ACS Publications [Internet]. 2021 Oct 13 [cited 2025 Feb 25];121(19):11971–2015. Available from: https://pubs.acs.org/doi/abs/10.1021/acs.chemrev.0c01174
Dean K, biology APN chemical, 2014 undefined. Advances in fluorescence labeling strategies for dynamic cellular imaging. nature.comKM Dean, AE PalmerNature chemical biology, 2014•nature.com [Internet]. [cited 2025 Feb 25]; Available from: https://idp.nature.com/authorize/casa?redirect_uri=https://www.nature.com/articles/nchembio.155 6&casa_token=WWdjSxoAqdoAAAAA:nG4oqUji1wLsCHYrX598G3lsNllMkiTY43vHR1KSfQ8WkJ 9zbcnirKSlJb799O_zTXI85BEsy_3NyEXROw
Chang W, Tan C, Sanjna †, Nerurkar N, Hai †, Cai Y, et al. Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy. Wiley Online LibraryWCC Tan, SN Nerurkar, HY Cai, HHM Ng, D Wu, YTF Wee, JCT Lim, J Yeong, TKH LimCancer Communications, 2020•Wiley Online Library [Internet]. 2020 Apr 1 [cited 2025 Feb 25];40(4):135–53. Available from: https://onlinelibrary.wiley.com/doi/abs/10.1002/cac2.12023
Harms, P. W., Frankel, T. L., Moutafi, M., Rao, A., Rimm, D. L., Taube, J. M., ... & Pantanowitz, L. (2023). Multiplex immunohistochemistry and immunofluorescence: a practical update for pathologists. Modern Pathology, 36(7), 100197.

Details

Primary Language

English

Subjects

Histology and Embryology

Journal Section

Research Article

Authors

Kubilay Doğan Kılıç ^*
0000-0002-9484-0777
Türkiye

Ayşegül Taşkıran Özyazıcı
0000-0001-9780-6948
Türkiye

Büşra Horuz
0009-0009-6203-4111
Türkiye

Publication Date

March 12, 2025

Submission Date

December 10, 2024

Acceptance Date

January 6, 2025

Published in Issue

Year 2025 Volume: 64 Number: 1

DOI

https://doi.org/10.19161/etd.1599016

IZ

https://izlik.org/JA34PY38DB

Cite

RIS / Bibtex

Vancouver

1.Kubilay Doğan Kılıç, Ayşegül Taşkıran Özyazıcı, Büşra Horuz. Antibody labeling kits vs conventional labeling: A comparative study in immunofluorescence of cell cultures. EJM. 2025 Mar. 1;64(1):118-27. doi:10.19161/etd.1599016