Lymphocyte separation medium

Lymphocyte separation medium, aprotinin, dimethyl sulfoxide (DMSO) and solvents for HPLC and LC/MS were purchased from Thermo Fisher Scientific (Ottawa, Ontario, Canada). Dextran, adenosine deaminase, leupeptin and potassium phosphate were obtained from Sigma-Aldrich Canada (Oakville, Ontario, Canada). HBSS was purchased from Wisent Bioproducts (St-Bruno, Quebec, Canada). 19-OH-prostaglandin (PG) B₂, PGB₂, PGB₂-D₄ and 17-octadecynoic acid (17-ODYA) were purchased from Cayman Chemicals (Ann Arbor, Michigan, USA). PF-4708671 was obtained from Abcam (Cambridge, Massachusetts, USA) and LY2584702 from Selleckchem (Houston, Texas, USA). Thapsigargin was obtained from Tocris Bioscience (Ellisville. Missouri, USA). LTB₄ was a generous gift from Dr Louis Flamand (Université Laval, Québec City, Canada). Recombinant CYP4F3A and the NADPH regenerating system were purchased from Corning (Corning, New York, USA). Protease and phosphatase inhibitor cocktail tablets were purchased from Roche (Laval, Quebec, Canada). Primary (anti-phospho-S6 #2211 and anti-S6 #2317) and secondary antibodies were obtained from Cell Signaling (Danvers, Massachusetts, USA). The enhanced chemiluminescent (ECL) substrate was obtained from Millipore Canada Ltd (Toronto, Ontario, Canada).

For single-cell suspensions, thymus and spleen were crushed and filtered through 80μm stainless steel mesh (Sefar Ltd., UK) in fluorescence-activated cell sorting (FACS) buffer (phosphate-buffered saline (PBS) containing; 2% heat-inactivated fetal calf serum (FCS) (Life technologies); and 5 mM ethylenediaminetetraacetic acid (EDTA) (Life technologies)). For spleen, red blood cells were removed by gradient centrifiguation at 1600rpm for 25 min using 4 mL of Lymphocyte Separation Medium (Fischer). For IEL preparations, faecal material was flushed from lumen of small intestine with ice-cold PBS using a gavage needle. Fatty tissues, vasculature and Peyer’s patches were removed, followed by longitudinal opening and 60 min agitation in RPMI-1640 10% Newborn calf serum (NCS) (Life technologies) with 5 mM EDTA (Life technologies) at 37 °C. Cells were subsequently passed through an autoclaved column containing 0.7 g of nylon wool (Polysciences, USA), equilibrated with RPMI-1640 with 38 mM HEPES (Life technologies). IELs were enriched on a discontinuous Percoll gradient (40%/80% isotonic Percoll), before use and stained as described below.

Leukemia cell samples were isolated from patients’ peripheral blood using lymphocyte separation medium (Thermo Fisher Scientific) and incubated in suspension in the RPMI 1640 medium described above. Patient 1 had T-cell prolymphocytic leukemia (T-PLL), patient 2 had mixed phenotype acute leukemia and patient 3 had AML. Patient samples were collected after obtaining written informed consent, and all studies using these samples were performed under a protocol approved by the Institutional Review Board of the UT MD Anderson Cancer Center, in accordance with the Declaration of Helsinki.