Histopaque 11191

Following patient biopsy, whole BM aspirates were tiled on top of a ficoll bilayer column (Histopaque 10771, Histopaque 11191—Sigma Aldrich, St. Louis, MO, USA). After centrifugation, the low-density (upper) and high-density (lower) layers were isolated and washed with PBS. Optical microscopy and flow cytometry allowed characterization of the layers’ cellular contents. The high-density layer contained myeloid lineage cells (morphologic characterization: promyelocytes 1.25%, myelocytes 7.75%, metamyelocytes 5.5%, band cells 5.5%, and neutrophils 80%). The percentages did not differ significantly between the control and the MDS group (p > 0.05, Mann–Whitney test). CD34+ were identified in the low-density layer, along with all other mononuclear BM cells. The low-density layer was used for CD34+ cell isolation using magnetic beads (Miltenyi Biotec, Auburn, CA, USA), per the manufacturer’s instructions.

Primary human tumor tissue samples paired together with peripheral tissues (also called “normal” or “healthy” of the same patients) were collected surgically from breast cancer patients treated at the Colchester General Hospital, following informed, and written consent taken before surgery. Paired normal (healthy) peripheral tissues were removed during macroscopic examination of a tumor by pathologists. Blood samples were collected before breast surgery from patients with primary breast cancer (PBC) and before treatment from patients with metastatic breast cancer (MBC). Samples were also collected from healthy donors (individuals with no diagnosed pathology), which were used as control samples. Blood separation was performed using buoyancy density method employing Histopaque 1119-1 (Sigma, St. Louis, MO) according to the manufacturer's protocol. Ethical approval documentation for these studies was obtained from the NRES Essex Research Ethics Committee and the Research & Innovation Department of the Colchester Hospitals University, NHS Foundation Trust [MH 363 (AM03) and 09/H0301/37].

Donor islets were isolated by a digestion method that described previously^{17 (link)}. The pancreas was perfused with 1 mg/ml collagenase P (Roche, Basel, Switzerland) and digested at 38 °C for 20 min. After digestion, the pancreatic tissue was mixed with Histopaque-10771 and Histopaque-11191 (Sigma-Aldrich), and the purified islet cells were obtained by density gradient centrifugation.

After being anesthetized with chloral hydrate (50ul/10g of 0.1g/ml solution), the common bile duct of mice was cannulated and injected of collagenase P (1mg/ml; Roche Diagnostics, Mannheim, Germany) in cold Hanks’ balanced salt solution (HBSS). The distended pancreas was excised and digested at 37°C for 10 min, and then shaken by hand for 10 s until the solution appears homogeneous. The digested tissues were filtered through a 600 μm mesh and washed by cold HBSS with 10% fetal bovine serum (FBS). Islets were purified using the Histopaque purification method (Histopaque 11191 and 10771, Sigma Aldrich, Saint Louis, USA) [34 (link)]. Freshly isolated islets were plated equivalently on sterile six-well plates and cultured in RPMI-1640 medium containing 10% FBS depleted of exosomes, 1% (v/v) antibiotics (100 U/ml penicillin: 0.1 mg/ml streptomycin, Sigma Aldrich, Saint Louis, USA) and 11.1 mM glucose at 37°C, 5% CO₂. To induce islets injury in vitro, islets were treated with three cytokines cocktail (TNFα 10 ng/ml, IL-1β 5 ng/ml and IFNγ 100 ng/ml, R&D Systems, Minneapolis, USA) or 0.5 mM STZ (in 0.1M sodium citrate, pH 4.5) for 24 hours [35 (link)]. Islet survival was evaluated by acridine orange/propidium iodide (AO/PI) staining.

Blood from four 16-week-old WA elite pure line chickens (Hendriks Genetics, Boxmeer, The Netherlands) [22 (link)] was collected from wing veins by heparin tubes. Next, the blood was diluted with sterile PBS (#10010-015, Gibco, Invitrogen, Paisley, UK), placed on Histopaque-1.1191 (#1119, Sigma-Aldrich, Saint Louis, MO, USA) and centrifuged at 700× g for 40 min at RT. The interphase was collected and washed 3× with PBS at 250 g for 8 min at RT. The isolated PBMCs were resuspended in a complete culture medium (RPMI 1640, (#52400-025, Gibco, Invitrogen, Paisley, UK)) with the addition of 10% heat-inactivated chicken serum (#16110082, Gibco, Invitrogen, Paisley, UK), 1% penicillin-streptomycin (#15140122, Gibco, Invitrogen, Paisley, UK) and 1% L-glutamine (#25030024, Gibco, Invitrogen, Paisley, UK) and seeded in 96-well, flat bottom cell culture plate (#3596, Corning Inc., Corning, NY, USA) at 500,000 cells per well and stimulated with pokeweed mitogen (PWM) (#L9379, Sigma, Saint Louis, MO, USA) at 10 and 20 µg/mL for 24 h and 48 h at 41 °C, 5% CO₂. Cell-culture supernatant was collected and stored at −80 °C for further tests.