Preparation of HOCl-Oxidized Tumor Cell Lysates

We followed the protocol from a previous report to prepare HOCl-oxidized LL2 and EG7-OVA lysate [18 (link)]. Briefly, NaOCl reagent (Sigma-Aldrich) was diluted with DPBS (Dulbecco’s Phosphate Buffered Saline, Cellgro) and immediately added to the tumor cells to prepare a 0.7 μM HOCl solution. The final cell density was 1 × 10⁶ cells/ml in DPBS. The tumor cell suspensions were incubated at 37 °C for 1 h with gentle agitation every 30 min to induce oxidation-dependent tumor cell death. At the end of the 1 h treatment, the tumor cells were harvested, washed twice with DPBS (3 times the volume of DPBS was added to each volume of HOCl solution to ensure complete removal of HOCl), and subjected to 7 freeze-thaw cycles. For this, the TCLs were frozen in liquid nitrogen for ≥5 min and thawed completely at room temperature 7 times to completely rupture the tumor cells.