488 nm laser

To purify mCherry^+ve cells for subsequent immunoblotting, cells transfected with the Hsp-encoding bicistronic constructs were harvested with trypsin 48 h post-transfection. Samples were washed twice in PBS (pH 7.4; 5 min, 300 × g) and resuspended in fluorescence-activated cell sorting (FACS) buffer (25 mM HEPES, 1 mM EDTA, 0.5% w/v bovine serum albumin in PBS, pH 7.0). Cell clumps were removed by straining through a 40 µm nylon mesh before analysis on an S3e Cell Sorter equipped with a 561-nm laser (Bio-Rad Laboratories, Hercules, CA, USA). mCherry^+ve cells were sorted such that 300,000 cells were recovered.
To confirm that cells resolved in the iPop did indeed have inclusions, cells were transfected to express mFluc-EGFP and fixed in 1% (w/v) PFA in PBS (pH 7.4) for 30 min on ice. Samples were washed twice in PBS (5 min, 300 × g) and resuspended in FACS buffer. Cells were sorted on a FACSAriaII equipped with a 488-nm laser (BD Biosciences, San Jose, CA, USA) at the MWAC BRIL Flow Cytometry Facility, University of New South Wales.

Plasma interleukin-6 levels were analyzed using a mouse cytometric bead array inflammation kit (BD Biosciences, San Diego, CA, USA) on a FACSCalibur flow cytometer affixed with a 488-nm laser (BD Immunocytometry Systems, San Jose, CA, USA), according to the manufacturer’s protocol. Plasma PGE₂ levels were determined using commercially available purification kits and enzyme-linked immunosorbant assay kits (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturer’s protocols. Blood β-hydroxybutyrate (βHB) levels were measured enzymatically on day 21 using the Precision Xceed system (Abbott, Tokyo, Japan) to evaluate the production of ketone bodies and to investigate the correlation between ketone bodies and anti-tumor efficacy.