1640 medium

The human epithelial ovarian cancer cell line A2780 was obtained from the European Collection of Authenticated Cell Cultures (ECACC) (EC93112519–F0; KAC, Hyogo, Japan). A2780 cells were grown in Roswell Park Memorial Institute (RPMI)–1640 medium (FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with heat-inactivated 10% fetal bovine serum (BioWest, Nuailé, France) at a passage number from 8 up to 18. Cultures were maintained at 37 °C in a humidified atmosphere containing 5% CO₂.

Human prostate cancer cell lines, prostatic carcinoma-3 (PC-3) (low expression of PSMA), and lymph node carcinoma of the prostate (LNCaP) (high expression of PSMA) were obtained from the RIKEN Cell Bank (Tsukuba, Japan). Cells were maintained in a culture medium, Roswell Park Memorial Institute 1640 medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), supplemented with 10% heat-inactivated fetal bovine serum (Gibco) and 1% penicillin-streptomycin (FUJIFILM Wako Pure Chemical). The medium for LNCaP was supplemented with 1% sodium pyruvate (FUJIFILM Wako Pure Chemical) in a culture medium. Cells were seeded in 24-well plates (5 × 10⁴/well) and cultured for 2 days. After washing twice with phosphate-buffered saline (PBS) (−), the culture medium was changed to Hanks’ balanced salt solution (+). After treatment with [²¹¹At]PSMA1 or [²¹¹At]PSMA5 (approximately 30–50 kBq/well), cells were washed twice with PBS (–). After washing, all cells were lysed with 0.1 N sodium hydroxide, and the radioactivity of the cells was calculated using a 2480 Wizard² γ counter (Perkin Elmer, MA, USA). Protein levels were measured using a plate reader (MultiScan FC, Thermo Fisher) and the BCA Protein Assay Kit (FUJIFILM Wako Pure Chemical). Uptake (%uptake/mg protein) was compared between PC-3 and LNCaP cells at 30 min after incubation with [²¹¹At]PSMA1 or [²¹¹At]PSMA5.

Homemade kefir was provided by Nakagaki Consulting Engineer and Co., Ltd. (Osaka, Japan). Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco’s modified Eagle’s medium (DMEM), and human interleukin-2 were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents were of analytical grade.

The human extravillous trophoblast HTR8/SVneo cell line was cultured in a Roswell Park Memorial Institute 1640 medium (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), supplemented with 10% fetal bovine serum (Nichirei Biosciences, Inc., Tokyo, Japan) and 1% PSN (100 μg/mL penicillin, 100 μg/mL streptomycin, and 200 μg/mL neomycin; Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C in humidified air containing 5% CO₂ [28 (link)].

Human trophoblast-like cell line HTR8/SVneo cells (ATCC, CRL-3271) were cultured in a Roswell Park Memorial Institute (RPMI) 1640 medium (Wako, Osaka, Japan) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (MP Biomedicals, Santa Ana, USA) and 1% penicillin-streptomycin (Nacalai Tesque, Kyoto, Japan). For the luciferase reporter assay, human embryonic kidney cell line HEK293T cells (ATCC, CRL-11268) were cultured in Dulbecco's modified Eagle medium (DMEM, Wako, Osaka, Japan) with the same supplements as the RPMI 1640 medium. Both types of cells were grown in a 5% CO₂ humidified incubator at 37°C.

The human breast cancer BT-474 cell line was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). BT-474 cells were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum (Wako Pure Chemical Industries), and 1% penicillin/streptomycin (Wako Pure Chemical Industries) in a humidified atmosphere of 95% air and 5% CO₂ at 37 °C.