Abstract
Immunostaining is one of the advantageous methods for the qualitative analysis of cellular markers of interest. Immuno-stained cells are typically analyzed by fluorescence microscopy or flow cytometry. Flow cytometry has the advantage of being able to process large numbers of cells in a short time thus enhancing its quantitative capacity. The staining protocol typically includes fixation of cells followed by permeabilization, blocking procedures to reduce non-specific binding of the label, and staining with specific antibodies labeled directly or indirectly with fluorescence-tags. Important controls include staining with a relevant non-immune antibody to identify any non-specific imminent globally binding and measurements in the absence of any fluorescent tag to detect non-specific sources of fluorescence. The most common source of non-specific fluorescence is caused by autofluorescence of naturally occurring chemicals within the cells of interest. In this study, we found high levels of cellular autofluorescence in mouse embryonic fibroblasts, at levels that interfered with the detection of a number of cellular antigens using common fluorophores. This autofluorescence was detected in three of the four fluorescence channels restricting useful analysis to only one channel (red) on the instrument. The study highlights an important limitation to immunostaining techniques and reinforces the need for the use of a thorough set of controls to ensure specificity of quantitative analysis.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
Not applicable.
Code Availability
Not applicable.
References
Casas E, Vavouri T (2020) Mechanisms of epigenetic inheritance of variable traits through the germline. Reproduction 159(6):R251–R263. https://doi.org/10.1530/Rep-19-0340
Nilsson EE, Sadler-Riggleman I, Skinner MK (2018) Environmentally induced epigenetic transgenerational inheritance of disease. Environ Epigenetics 4(2). https://doi.org/10.1093/eep/dvy016
Andersson H, Baechi T, Hoechl M, Richter C (1998) Autofluorescence of living cells. J Microsc-Oxford 191:1–7
Aubin JE (1979) Autofluorescence of viable cultured mammalian-cells. J Histochem Cytochem 27(1):36–43. https://doi.org/10.1177/27.1.220325
Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241:172–182
Li Y, O’Neill C (2012) Persistance of cytosine methylation of DNA following fertilisation in the mouse. Plos One 7(1):e30687
Celik S, Li Y, O’Neill C (2014) The exit of mouse embryonic fibroblasts from the cell-cycle changes the nature of solvent exposure of the 5 ‘-methylcytosine epitope within chromatin. PloS One 9(4):e92523. https://doi.org/10.1371/journal.pone.0092523
Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS (1986) Phenol Red in Tissue-Culture Media Is a Weak Estrogen - Implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci USA 83(8):2496–2500
Stadtfeld M, Varas F, Graf T (2005) Fluorescent protein-cell labeling and its application in time-lapse analysis of hematopoietic differentiation. Methods Mol Med 105:395–412. https://doi.org/10.1385/1-59259-826-9:395
Loike J, Silverstein S (1983) A fluorescence quenching technique using trypan blue to differentiate between attached and ingested glutaraldehyde-fixed red blood cells in phagocytosing murine macrophages. J Immunol Methods 57:373–379
Shilova ON, Shilov ES, Deyev SM (2017) The effect of trypan blue treatment on autofluorescence of fixed cells. Cytom Part A 91a(9):917–925. https://doi.org/10.1002/cyto.a.23199
Wallrabe H, Svindrych Z, Alam SR, Siller KH, Wang TX, Kashatus D, Hu S, Periasamy A (2018) Segmented cell analyses to measure redox states of autofluorescent NAD(P)H, FAD & Trp in cancer cells by FLIM. Sci Rep-UK 8. https://doi.org/10.1038/s41598-017-18634-x
Surre J, Saint-Ruf C, Collin V, Orenga S, Ramjeet M, Matic I (2018) Strong increase in the autofluorescence of cells signals struggle for survival. Sci Rep-UK 8. https://doi.org/10.1038/s41598-018-30623-2
Chacko JV, Eliceiri KW (2019) Autofluorescence lifetime imaging of cellular metabolism: Sensitivity toward cell density, pH, intracellular, and intercellular heterogeneity. Cytom Part A 95a(1):56–69. https://doi.org/10.1002/cyto.a.23603
Uzuner SC (2020) Mitochondrial DNA methylation misleads global DNA methylation detected by antibody-based methods. Anal Biochem 601. https://doi.org/10.1016/j.ab.2020.113789
Acknowledgements
Authors thank to Mr. Lindsay Peters for advice and assistance with flow cytometry calibrations and experiments.
Funding
This work was supported by a grant from the Australian National Health and Medical Research Council to Prof Chris O’Neill, and a Turkish Government Postgraduate Scholarship to Dr. Selcen Celik-Uzuner.
Author information
Authors and Affiliations
Contributions
SCU performed experiments. SCU and CO designed the study and wrote the manuscript.
Corresponding author
Ethics declarations
Conflict of Interest
Authors declare that there are no conflicts of interest.
Ethics Approval
Not applicable.
Consent to Participate
Not applicable.
Consent for Publication
Not applicable.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Çelik-Uzuner, S., O’Neill, C. Cellular Autofluorescence in Mouse Embryonic Fibroblasts Interferes with Antigen Detection Using Flow Cytometry. J Fluoresc 31, 873–879 (2021). https://doi.org/10.1007/s10895-021-02724-1
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10895-021-02724-1