Ficoll paque plus density gradient centrifugation

BM was flushed from the shafts of femurs and tibias from 6‐ to 8‐week‐old CD45.1⁺ C57BL/6 congenic mice, and single‐cell suspensions were prepared by passaging through a 70‐μm nylon cell strainer (Corning, Corning, NY, USA). Low‐density BM mononuclear cells (MNCs) were isolated by Ficoll‐Paque PLUS density gradient centrifugation (<1.077 g/ml; GE Healthcare, Uppsala, Sweden) to exclude granulocytes and red blood cells. Lineage‐positive cells expressing CD3, CD4, CD8 (T cells), CD11b (myeloid cells), B220 (B cells), Gr‐1 (macrophage/monocytes), CD71, TER119 (erythroid cells) or NK1.1 (natural killer cells) were removed from the MNCs by incubation with a rat anti‐mouse monoclonal antibody (mAb) cocktail as above (BD Biosciences, Franklin Lakes, NJ, USA), followed by incubation with sheep anti‐rat IgG‐conjugated magnetic beads (Dynal Inc., Oslo, Norway) with gentle agitation according to the manufacturer's protocol. Bead‐bound cells were removed using a magnetic particle concentrator (Dynal Inc.). The remaining lineage‐negative MNCs were considered as HSC‐eBMCs.

THP-1 acute monocytic leukemia ATCC TIB-202™ (short THP-1) cells were cultured in RPMI complete medium (RPMI 1640 medium (Gibco/Life Technologies) supplemented with 10% fetal bovine serum gold (FCS, PAA Laboratories), 2 mM L-glutamine, 50 units/ml penicillin and 50 µg/ml streptomycin (all Gibco/Life Technologies)) at 37°C in a humid 7.5% CO₂ atmosphere. Cells were maintained in culture by centrifugation at 125xg for 10 minutes and subsequently resuspended twice per week to a concentration of 200,000 viable cells/ml.
For isolation of Peripheral Blood Mononuclear Cells (PBMCs), buffy coats were produced from whole blood donations by using the Top & Bottom Extraction Bag System (PolymedMedical Devices). PBMCs were isolated from buffy coats by Ficoll^® Paque PLUS density gradient centrifugation (GE Healthcare GmbH). PBMCs were rested overnight in RPMI complete medium at 37°C in a humid 7.5% CO₂ atmosphere. PBMCs with suitable MAIT cell numbers (MAIT cells being >3% of CD3⁺ T cells) were stained for CD3, CD161, and TCR Vα7.2 for 15 minutes at 4°C. MAIT cells were sorted as CD3⁺ Vα7.2⁺ CD161⁺⁺ lymphocytes. Sorted cells were washed with FACS buffer (2% FBS (v/v), 2 mM EDTA in PBS) and rested overnight in RPMI complete medium at 37°C in a humid 7.5% CO₂ atmosphere.

NB4 and HL60 cell lines were purchased from DSMZ (Braunschweig, Germany). Bone marrow sample was obtained from a patient diagnosed with APL (promyelocytes consisted of 70% of bone marrow karyocytes; PML-RARA translocation was detected). White mononuclear cells were purified from bone marrow aspirate by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare Chicago, IL, USA). Ethical permission from Vilnius Regional Biomedical Research Ethics Committee (approval no. 158200-16-824-356) and informed consent of the patients were obtained. NB4 cells and freshly purified APL patient cells were seeded at density 0.5 × 10⁶ cells/ml and cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco, Carlsbad, CA, USA) at 37°C in a humidified 5% CO₂ atmosphere.

Peripheral blood mononuclear cells (PBMCs) from CAD patients and control individuals were isolated by Ficoll-Paque PLUS density gradient centrifugation (GE Healthcare Bioscience). The PBMCs were cryopreserved for less than 3 months at −80 °C in 90% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich) and 10% DMSO (Sigma-Aldrich). Monocytes were isolated using the EasySep™ Human Monocytes Isolation Kit (Stemcell Technology) and cultured in RPMI-1640 (Lonza) containing 2 mM glutaMAX (Gibco), 20 µg/mL gentamicin (Gibco), and 2% normal human serum type AB (Invitrogen).