Csu confocal spinning disk unit

MRL/N-1 fibroblastoid cells established from the spleens of MRL/MpTn-gld/gld mice (46 (link)) were incubated with 10 μM AF633-P140 for 4 h followed by staining with 100 nM LysoTracker Green at 37°C for 5 min, or cells were incubated with 10 μM AF488-P140 for 4 h followed by staining with 50 nM MitoTracker DeepRed at 37°C for 20 min. Stained cells were washed three times with phosphate-buffered saline (PBS) pH 7.4, and imaged immediately with a spinning-disc confocal microscope consisting of a CSU confocal spinning disk unit (Yokogawa), an EMCCD Evolve camera (Roper Scientific), mounted on an Axio Observer Z1 microscope (Zeiss) at 37°C with 5% CO₂ supply. Both LysoTracker Green and MitoTracker DeepRed were purchased from ThermoFisher Scientific.

After treatment, cells were fixed and permeabilized with 100% (v/v) pre-cooled methanol at −20 °C, followed by incubation with phycoerythrin (PE)-labeled antibody to HSPA8 (Abcam, ab65170) overnight at 4 °C. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were then mounted with mounting medium (DAKO) and subjected to fluorescence imaging using a spinning-disc confocal microscope consisting of a CSU confocal spinning disk unit (Yokogawa), an Electron Multiplying Charge Coupled Device (EMCCD) Evolve camera (Roper Scientific), mounted on an Axio Observer Z1 microscope (Zeiss). 405 and 561 nm lasers, 420–470 nm band pass and 570–640 nm band pass emission filters were used to image the DAPI and HSPA8-PE, respectively. Quantification of fluorescence signals was done as described in Fig. S1B in using Image J software (National Institutes of Health).