Stadtfeld M, Varas F, Graf T (2005) Fluorescent protein-cell labeling and its application in time-lapse analysis of hematopoietic differentiation. īerthois 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. Plos One 7(1):e30687Ĭelik 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. Li Y, O’Neill C (2012) Persistance of cytosine methylation of DNA following fertilisation in the mouse. Santos F, Hendrich B, Reik W, Dean W (2002) Dynamic reprogramming of DNA methylation in the early mouse embryo. J Microsc-Oxford 191:1–7Īubin JE (1979) Autofluorescence of viable cultured mammalian-cells. Īndersson H, Baechi T, Hoechl M, Richter C (1998) Autofluorescence of living cells. Nilsson EE, Sadler-Riggleman I, Skinner MK (2018) Environmentally induced epigenetic transgenerational inheritance of disease. 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.Ĭasas E, Vavouri T (2020) Mechanisms of epigenetic inheritance of variable traits through the germline. This autofluorescence was detected in three of the four fluorescence channels restricting useful analysis to only one channel (red) on the instrument. 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. The most common source of non-specific fluorescence is caused by autofluorescence of naturally occurring chemicals within the cells of interest.
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 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. Flow cytometry has the advantage of being able to process large numbers of cells in a short time thus enhancing its quantitative capacity. Immuno-stained cells are typically analyzed by fluorescence microscopy or flow cytometry.
Immunostaining is one of the advantageous methods for the qualitative analysis of cellular markers of interest.