Mcilwain tissue chopper

Detailed procedures for tissue transplantation have been previously described by Usai et al. [23 (link)]. Briefly, bulk tumor tissue screened by the histopathologist (at the Division of Surgical Pathology, University of Pisa) was washed three times with RPMI supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin and 2.5 µg/mL amphotericin; then, it was minced, firstly with a scalp blade (1–3 mm), and then using the McIlwain tissue chopper (Campden Instruments LTD, Loughborough, UK) to obtain pieces of about 0.3 mm × 0.3 mm × 0.3 mm. The pieces were stained with 10 µg/mL CM-Dil (Invitrogen, Carlsbad, CA, USA) in D-PBS and incubated for 30 min at 37 °C. Tissue pieces were then washed and centrifuged three times by D-PBS and resuspended in D-PBS supplemented with 10% FBS (Gibco, Waltham, MA, USA). Pieces of fluorescent-labeled tissue were manually transplanted into the perivitelline space of n = 90 AB wild-type recipient embryos 2 days post fertilization (dpf), which were lying in 1% agarose disks in multi-well plates. After transplantation, embryos were incubated for 2 h at 35 °C. The pool of embryos xenografted with the tissue derived from each patient will be hereinafter referred to as zPDXs.

After kill, brains were cut into 5 coronal slices of 2 mm thickness by using a McIlwain tissue chopper (Campden Instruments Ltd.) and incubated in 2% solution of 2,3,5‐triphenyltetrazolium chloride (TTC; Sigma‐Aldrich). Infracted area and volume were calculated from digital images (Canon 4×, Canon Inc.) and ImageJ software 36. The measure of lesion volume and area was performed on coronal brain slices for a total of three slices per animal.

Fresh NSCLC tissue and autologous peripheral blood was obtained from patients undergoing surgical resection through the Wales Cancer Bank. Samples were obtained under informed consent and with ethical approval (see above). Samples included in this report were confirmed as NSCLC by a certified histopathologist. Patient demographics corresponding to samples used in this study are shown in Table 1. PBMCs were isolated from fresh peripheral blood (10–20 mL) and CD14⁺ PBMCs isolated by positive selection as described above. Fresh tumour tissues were dissected into ~1mm³ explants using a McIlwain Tissue Chopper (Campden Instruments Ltd.; United Kingdom). CD14⁺ PBMCs (1 × 10⁵) were antibody stained and analysed by flow cytometry prior to culture to determine the baseline (T = 0) myeloid phenotype. CD14⁺ PBMCs (1 × 10⁵/well) were co-cultured with 5 explants/well of a 48-well flat-bottom cell-repellent surface plate (Greiner Bio-One; Austria) for 48 h in 1% FBS-containing complete RPMI-1640. M1-like, M2-like, and unpolarised media control Mφs were established in parallel. Explant only wells were setup to assess the in situ TAM phenotype. Explant-induced Mφ polarisation was assessed by flow cytometry. Viable myeloid cells were gated based on positive CD14 staining and negative Fixable Viability Dye eFluor780 staining.

Brains were removed and immersed into ice-cold (4°C) artificial cerebrospinal fluid (ACSF) with the following composition (in mM): 126 NaCl, 3.5 KCl, 2 CaCl₂, 1.3 MgCl₂, 1.2 NaH₂PO₄, 25 NaHCO₃, and 11 glucose, pH 7.4 equilibrated with 95% O₂ and 5% CO₂. Hippocampal slices (600 μm thick) were cut with a McIlwain tissue chopper (Campden Instruments, Ltd.) and kept in ACSF at 25°C. Slices were then transferred to a submerged recording chamber perfused with oxygenated (95% O₂ and 5% CO₂) ACSF (3 ml/min) at 34°C.