KML1, a cell line established from the pleural effusion of a patient with diffuse large B cell lymphoma (DLBCL), and A4/Fuk, a cell line established from the ascites fluid of a patient with DLBCL, were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition (JCRB1347 and JCRB0097, respectively). HF is another malignant lymphoma cell line, and was established from the pleural effusion of a patient with DLBCL; this line was purchased from American Type Culture Collection (CRL3383). Cryopreserved lymphoma cell lines were thawed and cultured in RPMI 1640 supplemented with foetal bovine serum (FBS; 10% for KML1 and A4/Fuk, and 15% for HF) for 2 days before observation by Raman spectroscopy. As control cells, peripheral B cells, activated T cells, neutrophils, and activated macrophages were used. Peripheral B cells, T cells, neutrophils, and macrophages were isolated from peripheral blood of 3 healthy volunteers (Volunteers A, B, and C) using the EasySep™ Human Pan-B Cell Enrichment Kit (Veritas, ST-19554), the EasySep™ Direct Human T Cell Isolation Kit (Veritas, ST-19661), the EasySep™ Direct Human Neutrophil Isolation kit (Veritas, ST-19666), and the EasySep™ Human Monocyte Isolation Kit (Veritas, ST-19359). These procedures were performed in accordance with an ethical research proposal approved by Medical Research Ethics Committee, Tokyo Medical and Dental University (M2017-292). Written informed consent was obtained from each donor. All experiments were performed in accordance with the tenets of the Declaration of Helsinki. Isolated B cells were maintained in RPMI 1640 supplemented with 10% FBS, and these cells were evaluated by Raman spectroscopy on the day of isolation. Isolated T cells were further activated by 1-week culturing in RPMI 1640 supplemented with 10% FBS along with Dynabeads Human T-Activator CD3/CD28 (Veritas, DB11131) and IL-2 (Corning, 354,043) in a 37 °C, 5% CO2 atmosphere. Isolated macrophages were activated according to the manufacturer’s 6-day culture protocol using ImmunoCult™-SF Macrophage Differentiation Medium (STEMCELL, 10,961) supplemented with 50 ng/mL macrophage colony stimulating factor, 10 ng/mL lipopolysaccharides, and 50 ng/ mL interferon gamma. Lymphoma cell lines, B cells, T cells, and macrophages were transported to the confocal Raman microscope in the respective culture media using a live-transport device and constant-temperature transport box (Sanplatec, iP-TEC®). Since neutrophils are fragile, cells of this type were isolated at the place of observation and were evaluated promptly. Full paper
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