H3667

Cells were grown on 13 mm coverslips in 12-well plates, washed in DPBS (14190136, Gibco™) before fixation with 4% paraformaldehyde and storage in DPBS. Before immunocytochemical staining, cells were washed in DPBS, and blocked using ADST [antibody diluent solution—triton: DPBS, 0.1 M L-Lysine (303341000, Thermo Scientific™), 1% w/v Human Serum Albumin Fraction V (12668-10GM, Sigma-Aldrich), Triton X-100 (A16046.AP, Thermo Scientific Alfa Aesar)] and 5% human serum (H3667, Sigma Aldrich) for 30 min. Cells were washed and primary antibodies at 2.5 µg/ml (suspended in ADST with 2% human serum) were applied overnight. After washing, secondary antibodies at 5 µg/ml and 4′,6-diamidino-2-phenylindole (DAPI, D1306, Invitrogen™) at 1 µg/ml (suspended in ADST with 2% human serum) were applied for 1 h, before mounting coverslips using Dako mounting medium (S302380-2, Agilent). Antibodies were sourced from Abcam: Rb anti-Ki67 (ab15580, ab16667), Ms anti-γH2AX (ab26350), Alexa Fluor ® 555 Goat pAb to Rb (ab150078, ab150086) and Alexa Fluor ® 488 Goat pAb to Ms (ab150117). Images were captured using the Leica DM4 B Upright Microsope at 10 × magnification and were counted manually using Leica Application Suite X 2019 3.7.1.21655v software (Leica Microsystems, Wetzlar, Germany).

MART-1 TCR CD8 T cells were generated as previously described.²¹ (link) In short, primary CD8 T cells were isolated from healthy male or female donor buffy coats (Sanquin, Amsterdam, the Netherlands), activated with plate-coated CD3 and CD28 antibodies (both 5 μg/ 2x10⁶ cells/ 24-well, 16-0037-85 and 16-0289-85, Thermo Fisher Scientific) for 48 h in primary human CD8 T cell medium (RPMI Medium (GIBCO) containing 10% human serum (H3667, Sigma-Aldrich), 100 U/ml of Penicillin-Streptomycin (GIBCO), 100 U/ml IL-2 (Proleukin, Novartis), 10 ng/ml IL-7 (11340077, ImmunoTools) and 10 ng/ml IL-15 (11340157, ImmunoTools)). Right after 48 h activation, T cells were removed from activation plates and spinfected with MART-1 TCR retrovirus on Retronectin-coated (25 μg/ 24-well, TB T100B, Takara) non-tissue culture-treated plates. Cells were harvested and maintained in primary human CD8 T cell medium 24 h after transduction. 1 week after transduction, MART-1 TCR expression was checked by flow cytometry (α-mouse TCR β chain, 553172, BD Pharmingen) and cells were cultured in human CD8 T cell medium (RPMI containing 10% fetal bovine serum (Sigma), 100 U/ml of Penicillin-Streptomycin (GIBCO) and 100 U/ ml IL-2 (Proleukin, Novartis)).

Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and according to national and international guidelines and has been approved by the authors' institutional review board. Human SCC tumors (n = 21) were obtained from SA Pathology under oversight of the Royal Adelaide Hospital Research Ethics Committee (approval number 120113). Human SCC serum (n = 3) samples were kindly donated by A/Prof Ian Darby, RMIT University, Victoria, Australia under oversight of the St Vincent's Hospital Research Ethics Committee (approval number HREC-A 129/09). The clinical investigations were conducted according to the Declaration of Helsinki principles and written informed consent was obtained. The control group for immunohistochemistry experiments consisted of human skin samples collected during cosmetic surgical procedures (ethics approval number 11-CHREC-F007) from four patients with no known dermatological conditions. Normal human serum (H3667) obtained from Sigma-Aldrich (Castle Hill, NSW, Australia) was used as a control for western blotting experiments using SCC patient serum.

30 samples derived from 24 patients with neuroblastoma were included in this study. All tumor samples were collected from patients at the Great Ormond Street Hospital following written informed consent. In the case of minors, consent was obtained from adults with parental responsibility, with written assent from the child being optional for older participants. Ethical approval for the study was obtained through the national United Kingdom research ethics application system of the UK Health Research Authority; reference 14/WM/1253 “Establishing primary cultures and cell lines from pediatric cancers”. Tumor tissue was disaggregated manually into 1–2 mm fragments in X-VIVO 15 media (BE02-060F, Lonza) supplemented with 5% heat-inactivated human AB serum (H3667, Sigma-Aldrich), 100IU/mL penicillin (P4333, Sigma-Aldrich) and 100 μg/mL streptomycin (P4333, Sigma-Aldrich).

For the Troponin I tests, we utilized commercialized kits (Humasis, Republic of Korea). CNTI recombinant antigen (GRNCTNIN101, Fappon) was employed as a control material and serially diluted using human serum (H3667, Sigma Aldrich). We prepared samples covering a broad concentration range, ranging from 0.5 ng/ml to 20 ng/ml (clinically relevant range: >0.4 ng/ml). Negative data were exclusively derived using the running buffer from each kit. Both positive and negative datasets were systematically captured as time-series at 10-s intervals.