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After 48-hour incubation at 37°C, the inoculum was removed by washing, cells were lysed with addition of luciferase cell culture lysis reagent (Promega) and the plates were stored at -80°C until assessed for luciferase activity, which was measured in relative light units (RLU).
The supernatants were collected and stored at −20°C until assessed.
Thereafter, the supernatants and cells were collected and stored until assessed.
Cells were rinsed three times with 10 mM PBS and stored at 4°C until assessed.
The flank tumours were excised, rapidly frozen (<1 min) in a pre-cooled isopentane freezing bath (−70°C), and stored in liquid nitrogen until assessed.
At the times specified after GV removal and rinsing, aliquots of culture media were harvested and stored at -20°C until assessed for their content of the selected cytokines.
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CEO of Professional Science Editing for Scientists @ prosciediting.com