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Volume 9
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Technical Note

A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells

by
Andrea González-Báez
1,
Raúl Rangel-López
2,
Gustavo Hernández-Vidal
1,
Armando Trejo-Chávez
1,
Luis Rodríguez-Tovar
1,
Diana Zárate-Triviño
2,
Diana Caballero-Hernandez
2,
Rodolfo Nino Fong
3,
Gerardo Méndez-Zamora
4 and
Diana Elisa Zamora-Avila
1,*
1
Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Nuevo León, San Nicolás de los Garza 66054, Nuevo Léon, Mexico
2
Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza 66455, Nuevo Léon, Mexico
3
Veterinary School, University of Long Island, New York, NY 11548, USA
4
Facultad de Agronomía, Universidad Autónoma de Nuevo León, San Nicolás de los Garza 66054, Nuevo Léon, Mexico
*
Author to whom correspondence should be addressed.
Methods Protoc. 2026, 9(5), 137; https://doi.org/10.3390/mps9050137 (registering DOI)
Submission received: 29 June 2026 / Revised: 10 September 2026 / Accepted: 10 September 2026 / Published: 20 September 2026
(This article belongs to the Section Molecular and Cellular Biology)
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Abstract

The 4T1 cell line is a widely used model for triple-negative breast cancer (TNBC) research due to its high metastatic potential and resemblance to clinical progression. However, its biological characteristics often limit the efficiency and reproducibility of conventional nucleic acid delivery protocols. Here, we present an optimized non-viral delivery framework for efficient cell-associated uptake in 4T1 cells. Five carrier systems—Xfect RNA polymer (Xfect), polyethyleneimine (PEI), chitosan (CHI), and gold nanoparticle conjugates (AuPEI and AuCHI)—were systematically evaluated using both standard (adherent) and reverse (suspension) delivery approaches. A fluorescently labeled oligonucleotide (5′-TYE563) served as a reporter cargo at final concentrations of 5 nM and 10 nM, comparing complexation times of 15 s and 15 min. The proposed protocol introduces a rapid complexation strategy combined with reverse delivery, significantly reducing preparation time while enhancing cell-associated uptake. Under optimized conditions (10 nM, 15 s, reverse), Xfect achieved near-complete cellular uptake with minimal cytotoxicity. Crucially, the colocalization of dual-fluorescence signals enabled the simultaneous validation of metabolic viability and internalized cargo within a single field of view, bypassing the need for independent bulk cytotoxicity assays. This methodology provides a rapid, reproducible, and cost-effective framework to standardize early-stage delivery conditions for difficult-to-transfect cell lines such as 4T1.
Keywords: Xfect; polyethylenimine; chitosan; cell-associated uptake; complexation; 4T1 cells; gold nanoparticles; cell viability Xfect; polyethylenimine; chitosan; cell-associated uptake; complexation; 4T1 cells; gold nanoparticles; cell viability
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MDPI and ACS Style

González-Báez, A.; Rangel-López, R.; Hernández-Vidal, G.; Trejo-Chávez, A.; Rodríguez-Tovar, L.; Zárate-Triviño, D.; Caballero-Hernandez, D.; Fong, R.N.; Méndez-Zamora, G.; Zamora-Avila, D.E. A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells. Methods Protoc. 2026, 9, 137. https://doi.org/10.3390/mps9050137

AMA Style

González-Báez A, Rangel-López R, Hernández-Vidal G, Trejo-Chávez A, Rodríguez-Tovar L, Zárate-Triviño D, Caballero-Hernandez D, Fong RN, Méndez-Zamora G, Zamora-Avila DE. A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells. Methods and Protocols. 2026; 9(5):137. https://doi.org/10.3390/mps9050137

Chicago/Turabian Style

González-Báez, Andrea, Raúl Rangel-López, Gustavo Hernández-Vidal, Armando Trejo-Chávez, Luis Rodríguez-Tovar, Diana Zárate-Triviño, Diana Caballero-Hernandez, Rodolfo Nino Fong, Gerardo Méndez-Zamora, and Diana Elisa Zamora-Avila. 2026. "A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells" Methods and Protocols 9, no. 5: 137. https://doi.org/10.3390/mps9050137

APA Style

González-Báez, A., Rangel-López, R., Hernández-Vidal, G., Trejo-Chávez, A., Rodríguez-Tovar, L., Zárate-Triviño, D., Caballero-Hernandez, D., Fong, R. N., Méndez-Zamora, G., & Zamora-Avila, D. E. (2026). A Quick Reverse Transfection and Dual-Imaging Protocol for Simultaneous Viability and Oligonucleotide Delivery Assessment in 4T1 Cells. Methods and Protocols, 9(5), 137. https://doi.org/10.3390/mps9050137

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