The procedures used in this experiment have been published previously and were applied in this study with minor modifications [35 (link)]. Briefly, 14 days following the DC immunization described in Section 2.13 (set as day 0), the mice subcutaneously received 2 × 105 EG7 in the back. In addition, some mice further received the following antibodies intraperitoneally on days −3, −1, 2, and 5, and every 7 days thereafter until day 28: mouse anti-CD8a monoclonal antibody (clone 2.43: Bio X Cell, Lebanon, NH, USA) at 150 μg/mouse for CD8+ T cell depletion, rabbit anti-asialo GM1 antibody (FUJIFILM Wako Pure Chemical Corporation, Tokyo, Japan) at 100 μL/mouse for NK cell depletion, and isotype control (clone LTF-2: Bio X Cell, Lebanon, NH, USA) at 150 μg/mouse. Flow cytometry confirmed that 97% of the CD8+ T cells and 99% of the NK cells in the peripheral blood were depleted.
The tumor size was measured twice in a week using a caliper. The tumor volume was calculated using the following formula: V = 1/2 (L × W2), where L = length and W = width. The mice were euthanized when the maximum tumor diameter exceeded 20 mm, or their body weight loss exceeded 20%.
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