Pcr mycoplasma detection kit

The establishment of tumor cell lines was performed as previously described (28 (link)). Briefly, we cultured 1 × 10⁷ digested tumor cells in RPMI1640, including 10% FBS (FBS; Thermo Fisher Scientific), 100 U/mL penicillin, 100 μg/mL streptomycin, 50 μg/mL gentamicin, and 0.25 μg/mL amphotericin B (Thermo Fisher Scientific). We passaged tumors at approximately 80%–90% confluent, which was used when free of fibroblasts and growing beyond 10 passages.
MC-38 cell line (mouse colon cancer) was purchased from Kerafast. NFAT-luciferase reporter Jurkat (NFAT-Luc-Jurkat) cell line was purchased from InvivoGen. Jurkat cell line was maintained in RPMI1640 medium (Thermo Fisher Scientific) and MC-38 cell line was maintained in DMEM (Thermo Fisher Scientific). Both were supplemented with 10% FBS. All tumor cells were used after confirming that they were negative for Mycoplasma using the PCR Mycoplasma Detection Kit (TaKaRa) according to the manufacturer's protocol.

293T, MT4 (RRID:CVCL_2632), HeLa, MAGIC5 (HeLa derivative [Mochizuki et al., 1999 (link)]), and HOS cells were maintained under standard conditions. Cells were originally obtained from ATCC (except MAGIC5 cells) and routinely tested negative for mycoplasma contamination (PCR Mycoplasma Detection kit, Takara).

The HEK293T (human embryonic kidney cell), CT26 (murine colon cancer), RENCA (murine renal cell carcinoma), and A20 (murine lymphoma) cell lines were purchased from ATCC (Manassas, VA). The HEK293T and CT26 cell lines were maintained in DMEM (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% foetal calf serum (FCS; Cytiva, Tokyo, Japan). The RENCA and A20 cell lines were maintained in RPMI medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% FCS. All tumour cells were confirmed to be Mycoplasma (−) using a PCR Mycoplasma Detection Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions before use. Recombinant human Wnt3a protein was obtained from R&D (Minneapolis, MN). Recombinant murine Wnt3a protein was purchased from PeproTech (Cranbury, NJ). Rat anti-mouse PD-1 mAb (RMP1-14) and control rat IgG2a mAb (RTK2758) were obtained from BioLegend (San Diego, CA).

A tissue sample was obtained from an American miniature horse that had died of natural causes in a private Zoo (World Ranch, Osaka, Japan). The 15-year-old male and 13-year-old male individuals were registered by The American Miniature Horse Association as a pet. Muscle fragments were obtained from a syringe injected into the hind leg muscles and primary cells were established by explant culture in high glucose DMEM supplemented with 10% fetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin-amphotericin mixture and 1% Zellshield (Minerva Biolabs, Berlin, Germany) at 37 °C in a CO₂ incubator for 10 days. After several passages by 0.25% trypsin-1 mM EDTA solution, the primary cells were suspended in CultureSure cell freezing medium in a liquid nitrogen tank until use. To confirm mycoplasma contamination, established cells were subjected to PCR Mycoplasma Detection kit (Takara Bio, Shiga, Japan).

PK15 is an established cell line that originated from kidney epithelial cells of an adult pig (Sus scrofa domestica) (Todaro et al., 1974 (link)), and was generously provided by Dr. Yolanda Revilla (CBMSO, Madrid, Spain). Cells were maintained in low-glucose Dulbecco’s modified Eagle medium (DMEM) (Life Technologies, Paisley, UK) supplemented with 5% fetal bovine serum (FBS), glutamine (2 mM), penicillin (50 U/mL) and streptomycin (50 μg/mL) (Gibco, CA, USA), at 37 °C in a humidified atmosphere of 5% CO₂. The absence of mycoplasma was confirmed by using the PCR Mycoplasma Detection kit (Takara Bio, San José, CA, USA).

To establish tumor cell lines, 1×10⁷ digested tumor cells were cultured in RPMI1640, containing 10% fetal bovine serum (FBS; Cytiva, Tokyo, Japan), penicillin, streptomycin, and amphotericin B (Thermo Fisher Scientific). Tumor cells were passaged at approximately 80%–90% confluence and used when free of fibroblasts and proliferating beyond the 10th passage. The MEL01 cell line was generated from a previously reported patient with melanoma who acquired resistance after an initial response to PD-1 blockade.23 (link) This cell line lost the B2M gene and had no MHC class I expression.23 (link) Both MEL02 and MEL03 cell lines were generated from melanoma super-responders to PD-1 blockade before the initiation of therapy. The MEL04 cell line was generated from a patient with melanoma who acquired resistance to anti-PD-1 and anti-CTLA-4 mAbs after initial response to anti-PD-1 mAb (online supplemental table S1 and figure S1).
B16F10 (mouse melanoma) and EMT6 (mouse breast cancer) cell lines were purchased from ATCC (Manassas, Virginia, USA) and were maintained in the RPMI1640 medium supplemented with 10% FBS. All tumor cells were used after confirming that they were Mycoplasma (−) after Mycoplasma testing with the PCR Mycoplasma Detection Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions.