Nk 92mi

The human thyroid cancer cell line CAL-62 was purchased from the American Type Culture Collection (Rockville, MD, USA). The thyroid cancer cells were maintained in DMEM/high-glucose medium (HyClone Laboratories, Inc., South Logan, UT, USA) supplemented with 10% FBS and 100 U/ml penicillin/streptomycin. The NK-92MI human NK cell line was obtained from the American Type Culture Collection. NK-92MI cells were incubated in stem cell growth medium (Cellgro, Freiburg, Germany) supplemented with 2% human serum and 100 U/ml penicillin/streptomycin. CAL-62 cells were transduced with lentivirus co-expressing the Rluc (Renilla luciferase), mcherry, and puromycin genes under the control of the CMV promoter (Genecopoeia, Rockville, MD, USA). Stable clones were selected with puromycin (Sigma-Aldrich, St. Louis, MO, USA). CAL-62 and NK-92MI cells were retrovirally transduced to express both the effluc (enhanced firefly luciferase) and Thy1.1 genes. Thy1.1-positive cells were sorted by CD90.1 microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). The established stable cell lines expressing the Rluc and effluc genes were referred to as the CAL-62/R, CAL-62/F, and NK/F cells.

The human glioblastoma cell line U87/MG and human NK cell line NK92-MI were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). U87/MG cells were cultured in RPMI 1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA) and 1% penicillin–streptomycin (Hyclone). NK92-MI cells were cultured in stem cell growth medium (CellGro, Freiburg, Germany) supplemented with 2% exosome-depleted human serum (ultracentrifuged at 100,000 × g for 18 h) and 1% penicillin–streptomycin, at 37°C in 5% CO₂. U87/MG cells were transfected with a recombinant retrovirus containing a plasmid that showed enhanced expression of firefly luciferase (effluc) and thy1.1 genes, driven by a long terminal-repeat promoter (Retro–LTR–effluc–thy1.1). Thy1.1-positive cells were sorted from U87/MG cells expressing both effluc and thy1.1 genes using a magnetic cell sorter (Miltenyi Biotec, Bergisch Gladbach, Germany). Reverse transcription polymerase chain reaction (RT-PCR) and western blotting were performed to confirm the expression of effluc mRNA and protein, respectively. Established stable cells expressing both effluc and thy1.1 genes were referred to as U87/MG/F cells.