Table 3 List of recommendations based on the different parameters
From: Pitfalls in methods to study colocalization of nanoparticles in mouse macrophage lysosomes
Parameter | Recommendation |
|---|---|
LysoTracker probe | Check for intensity overlap of LysoTracker probe with the NP fluorescence properties Do not exceed maximal incubation time Perform imaging of LysoTracker probes in cell culture medium without phenol red Perform a pilot study and cell viability test to acquire the time range wherein your cell’s viability remains acceptable Do not exceed this time scale for continuous live-cell imaging (e.g., for J774.A1: 8 h) For experiments exceeding this time range, conduct a staggered set-up and stain with LysoTrackers before imaging |
Particle probe | Use the same fluorophore marker when comparing particles of different sizes |
CLSM Imaging | Apply optimal imaging parameters considering optimal resolution of sample to proceed with the colocalization analysis, as bias will increase with decreasing resolution Optimize the pinhole setting Never use (maximum) intensity projections Avoid noise and saturation events If available, use photon-counting detectors |
Nanoparticle-lysosome colocalization analysis | Describe data with adequate colocalization coefficients to provide the precise information about NP intracellular fate Include the cytofluorograms Analyse enough cells e.g. eight to ten single cells in the entire z-stack for each experimental condition, in case of high variability the number has to be increased Perform analysis on single cells selected from region of interest (ROI) to avoid noise contribution For colocalization analysis 3D data is recommended to be used. Keep in mind the different NP uptake kinetics and variability among cell lines Adjust experimental set-up specifically to the research question |
Data analysis | PCC does not provide detailed information about the distribution of NP arriving into lysosomes and the fraction of remaining or newly formed empty lysosomes Manders’ correlation coefficients M1 and M2 provide information on the fraction of the NP co-occurring with lysosomes within the resolution of a single voxel |