10.6084/M9.FIGSHARE.C.6273780
Aura Maria Moreno-Echeverri
Aura Maria
Moreno-Echeverri
Adolphe Merkle Institute
Eva Susnik
Eva
Susnik
Adolphe Merkle Institute
Dimitri Vanhecke
Dimitri
Vanhecke
Adolphe Merkle Institute
Patricia Taladriz-Blanco
Patricia
Taladriz-Blanco
International Iberian Nanotechnology Laboratory
Sandor Balog
Sandor
Balog
Adolphe Merkle Institute
Alke Petri-Fink
Alke
Petri-Fink
Adolphe Merkle Institute
University of Fribourg
Barbara Rothen-Rutishauser
Barbara
Rothen-Rutishauser
Adolphe Merkle Institute
Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes
Abstract Background In the field of nanoscience there is an increasing interest to follow dynamics of nanoparticles (NP) in cells with an emphasis on endo-lysosomal pathways and long-term NP fate. During our research on this topic, we encountered several pitfalls, which can bias the experimental outcome. We address some of these pitfalls and suggest possible solutions. The accuracy of fluorescence microscopy methods has an important role in obtaining insights into NP interactions with lysosomes at the single cell level including quantification of NP uptake in a specific cell type. Methods Here we use J774A.1 cells as a model for professional phagocytes. We expose them to fluorescently-labelled amorphous silica NP with different sizes and quantify the colocalization of fluorescently-labelled NP with lysosomes over time. We focus on confocal laser scanning microscopy (CLSM) to obtain 3D spatial information and follow live cell imaging to study NP colocalization with lysosomes. Results We evaluate different experimental parameters that can bias the colocalization coefficients (i.e., Pearson’s and Manders’), such as the interference of phenol red in the cell culture medium with the fluorescence intensity and image post-processing (effect of spatial resolution, optical slice thickness, pixel saturation and bit depth). Additionally, we determine the correlation coefficients for NP entering the lysosomes under four different experimental set-ups. First, we found out that not only Pearson’s, but also Manders’ correlation coefficient should be considered in lysosome-NP colocalization studies; second, there is a difference in NP colocalization when using NP of different sizes and fluorescence dyes and last, the correlation coefficients might change depending on live-cell and fixed-cell imaging set-up. Conclusions The results summarize detailed steps and recommendations for the experimental design, staining, sample preparation and imaging to improve the reproducibility of colocalization studies between the NP and lysosomes. Graphical Abstract
Biophysics
Biochemistry
Space Science
29999 Physical Sciences not elsewhere classified
Medicine
Cell Biology
Biotechnology
figshare
2022
2022-10-31
2022-10-31
Collection
10.1186/s12951-022-01670-9
CC BY 4.0