Lymphoprep density gradient medium

PBMCs were isolated from the blood of healthy donors and patients with PDAC using Lymphoprep™ density gradient medium in Sepmate™-50 tubes (StemCell Technologies). Isolated PBMCs were immediately frozen in FBS with 10% DMSO until further use. Samples were obtained under the approval of the Oxford Radcliffe Biobank research tissue bank ethics and after informed, written consent of the patients.

Blood was recruited from healthy donors at St. Olavs Hospital HF, the Bloodbank (project approved by Regional Ethical Committee of Mid-Norway; #2016/553). Peripheral blood mononuclear cells (PBMC) were isolated using SepMate separation tubes with LymphoPrep density gradient medium from STEMCELL Technologies (Cambridge, UK), according to the manufacturer’s recommendations. For experiments, 1 x 10⁶ cells per well were plated in 1 mL Roswell Park Memorial Insitute (RPMI) medium supplemented with 5% fetal bovine serum (FBS), 0.3 mg/mL glutamine, and 0.1 mg/mL gentamicin. Inhibitors were added 2 h prior to the addition of the Ca⁺⁺ ionophore A23178 (30 µM, 15 min) to activate cPLA₂α or lipopolysaccharide (LPS) (10 ng/mL, 72 h) as a potent inducer of inflammation. Following treatment, the cell suspensions were centrifuged to isolate the supernatant from the cell fraction. Samples were stored at −80 °C until analysis.

Blood was collected for peripheral blood mononuclear cell (PBMC) isolation (in S-Monovettes® K3 EDTA (Sarstedt) or equal), as well as for serum isolation (S-Monovettes® Serum (Sarstedt) or equal). The blood was diluted with an equal volume of PBS (Corning) containing 2% fetal bovine serum (FBS; Gibco; heat-inactivated; from Brazil). PBMCs were obtained by density gradient centrifugation using SepMate™-50 tubes (Stemcell Technologies) filled with Lymphoprep™ density gradient medium (Stemcell Technologies) according to the manufacturer’s instructions. After the isolation, the PBMCs were stained with Acridine Orange and DAPI (solution 18, Chemometec), counted using the NC-250 cell counter (Chemometec), frozen in FBS with 10% DMSO, and stored in liquid nitrogen for at least one week before further experiments. Blood collected for serum isolation was centrifuged 10 min at 2,000xg. Serum was transferred into Eppendorf tubes and frozen at -20°C until use for further experiments.

To isolate peripheral blood mononuclear cells, blood was collected in EDTA containing tubes and 1 serum tube. After dilution with DPBS (1 part blood; 3 parts DPBS), a ficoll density gradient was performed by using Lymphoprep density gradient medium (#07851, Stemcell technologies) and Leucosep tubes (#871346, OpoPharma) for 25 min (453 g, 22°C, brake: 1, acceleration: 4). The Lymphoprep layer was washed with FACS Buffer, and Red Cell Lysis Buffer (154mM NH₄Cl, 10mM KHCO₃, 0.1 mM EDTA) was used to remove residual erythrocytes. The remaining cells were used for further flow cytometry staining.

We isolated PBMCs from healthy donors and cryopreserved these cells to titrate and validate our antibody panel as well as to use in our single control and FMO compensation assays. Approximately 250 mL of healthy donor whole blood in EDTA tubes was acquired from Research Blood Products LLC Boston, delivered at RT. Upon delivery, whole blood was diluted 1:1 in 2% heat-inactivated fetal bovine serum (Hi-FBS) (Sigma, Saint-Louis, MO, USA, cat#F4135) in Hanks’ Balanced Salt Solution (HBSS), without calcium or magnesium (Thermo Fisher, Waltham, MA, USA, cat#14170112), i.e., washing buffer (WB). After careful mixing, the suspension was layered on top of an equal volume of Lymphoprep density gradient medium (Stem Cell, Vancouver, BC, Canada, cat#07811) in a 50-mL tube and centrifuged at 800× g for 20′ at RT with brakes off. Afterwards, using a sterile dropper pipet, the PBMC layer was collected in a new 50-mL tube and washed with WB. PBMCs were next centrifuged at 400× g for 10′ at RT with brakes on. The tube was decanted, flicked 3 times, and the PBMC pellet was resuspended in 50 mL WB. The tube was again centrifuged at 400× g for 10′ at RT, then resuspended in WB and counted before subsequent cryopreservation.

Peripheral blood mononuclear cells (PBMCs) were isolated from the buffy coat of healthy donors (Memorial Blood Centers in St. Paul, Minnesota) by density centrifugation using Lymphoprep density gradient medium (STEMCELL Technologies) and SepMate tubes (STEMCELL Technologies). T cells were isolated from PBMCs using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies).