Oil objective

Cells were grown on coverslips at low density in complete DMEM for 24 h. Cells were then fixed, permeabilized, and stained with Alexa Fluor 488 wheat germ agglutinin (WGA; W11261; Fisher, 1:1,000) to define cell boundaries, and then mounted on slides using ProLongGold antifade reagent with DAPI (P36931; Invitrogen) to visualize DNA. Cells were imaged on a Leica DMi8 inverted fluorescence microscope using a Leica 63×/1.4 oil objective, with an 8 × 8 tile scan captured using a Leica DFC9000GT digital camera and LASX acquisition software. The number of nuclei per cell was then manually quantified.

Neutrophils or monocytes were settled on poly-l-lysine–coated glass coverslips and cultured for 3 h, or for 60 min for CCCP and O+A experiments. Cells where then rinsed with PBS and fixed with 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilized with 0.05% Triton X-100 in PBS for 5 min at room temperature, and then treated with Blocking Buffer (1% goat serum, 1% FcR Blocking Reagent and 1% BSA in PBS) for 30 min at room temperature. Primary and secondary antibody stainings were performed in Staining Buffer (1% BSA in PBS). Isotype specific anti–mouse or anti–rabbit Alexa Fluor 488 or Alexa Fluor 568 were used as secondary antibodies. Counterstaining of cell nuclei was performed with the Hoechst stain (Molecular Probes). Samples were embedded in ProLong Gold Antifade Reagent (Molecular Probes) and examined with a TCS SP5 confocal laser-scanning microscope equipped with a 63×/1.4 oil objective (Leica). ImageJ software (Version 1.47t; National Institutes of Health) was used for analysis. For the TUNEL assay, the Click-iT TUNEL Imaging Assay kit (Molecular Probes) was used according to the manufacturer’s instructions. The percentage of co-localization was calculated from the Manders' Overlap Coefficient using the Co-localization analysis plugin (ImageJ).

LLPS analyses of p53 were performed using DIC and fluorescence microscopy tests under various conditions, including variations in temperature, concentration and molecular crowding. The samples (20 μL) in buffer A (50 mM Tris–Cl, pH 7.4, 150 mM NaCl and 5 mM DTT) were loaded onto a glass slide and cover slip system prepared as described previously.^{66 (link)} The images were acquired using a Leica TCS-SPE confocal microscope with a 63×/1.4 oil objective and a 488 nm laser. For fluorescence analysis, the samples were excited at 405 nm, and fluorescence signals were acquired at 450–500 nm. The images were processed using Fiji (a distribution package of ImageJ software, USA).