Model bx51tf

On day 24 after tumor inoculation, three mice in each group were sacrificed, and the tumors were removed. The tumor tissues were then fixed in 4% (w/v) formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). For immunohistochemical analysis, the sections were deparaffinized, followed by antigen retrieval and blocking. The sections were then stained with anti‐mouse CD3 mAbs (1:500 dilution; Abcam, Cambridge, MA, US). The histopathological and immunohistochemical sections were observed using a microscope (Olympus, Model BX51TF, America, Inc.).

The immortalized PAMs (2 × 10⁵ cells/well) were seeded in a six-well plate containing RPMI 1640 (Hyclone) with 15% FBS and 5 µg/mL gentamicin. The cells were trypsinized at 24, 48, 72, 96 h post-seeding, and then counted using the trypan blue exclusion method [43 (link)]. The number of cells at each time point was plotted. To measure cell anchorage-independent proliferation, which is a typical characteristic of immortalized cells, 1 × 10⁴ cells/well of iPAM 303 and iPAM61 were cultured in a six-well soft agar dish prepared with 0.8% agar and 0.7% agarose in the bottom and upper layers, respectively, for 2 weeks. Cells were fed with cell culture media every 2 days. Colony formation was checked under a light microscope (Model BX51TF, Olympus, Tokyo, Japan).

Cells were seeded on glass cover slips (Waner Instruments, LLC, Hamden, USA) in a six-well plate at a density of 1.2 × 10⁵ cells/well and cultured for 24 h. Subsequently, the cover slips were washed in 1 × phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min at room temperature. Cells were washed with 1× PBS and then incubated in 1× PBS with 5% bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) for 30 min. Subsequently, the cells were incubated with mouse anti-SLA-DRB1 monoclonal antibody (1:250 dilution, Bio-Rad, California, USA) in 1× PBS containing 2.5% BSA for 2 h. For visualization, cells were incubated with a goat anti-mouse antibody conjugated with Alexa Fluor 568 (1/500 dilution; Invitrogen, Massachusetts, USA) in 1× PBS containing 2.5% BSA for 1 h. For covering with a glass cover slip, VECTASHIELD Mounting Medium with DAPI (Vector, Burlingame, CA, USA) was used. Samples were analyzed under a fluorescence microscope (Model BX51TF, Olympus, Tokyo, Japan).