Ficoll hypaque density centrifugation

The AML cell lines THP-1, HL60, KG1 U937 and HEL were purchased from the American Type Culture Collection (Manassas, VA, USA). All cell lines were cultured in RPMI-1640 medium (Sigma, St Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco-Invitrogen, Carlsbad, CA, USA), L-glutamine, penicillin and streptomycin (Sigma) at 37 ^oC in 5% CO₂.
Refractory AML cells (patient 1: FAB M1, patient 2: FAB M4, patient 3: MDS-AML, patient 4: FAB M2, patient 5: FAB M0, patients 6: MDS-AML, patient 7: FAB M5a) were extracted from the bone marrow and peripheral blood after obtaining the appropriate informed consent. CD34⁺ AML cells were isolated from patient 3 with CD34 microbeads kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Patients with AML and two independent donors were examined as approved by the institutional review board at the Hiroshima University. These specimens including more than 90% of AML cells were subjected to Ficoll-Hypaque density centrifugation (GE Healthcare Bio-Sciences, Uppsala, Sweden).

PBMCs were isolated by Ficoll-Hypaque density centrifugation (GE Healthcare) from cytopheresis or whole-blood samples obtained from healthy volunteers and patients, respectively. PBMCs and EBV-B cells (purified B cells were immortalized with EBV in the laboratory) were cultured in RPMI medium supplemented with 10% FBS, whereas HEK293T cells (ATCC; CRL-3216) were cultured in DMEM medium supplemented with 10% FBS. Subsets were separated by MACS, using magnetic beads conjugated with the appropriate antibody (Miltenyi Biotec) according to the manufacturer’s protocol. All cells used in this study were tested for mycoplasma contamination and found to be negative.

ILC2s population was defined as Lin⁻ICOS⁺IL⁻17RB⁺. Heparinized venous blood was freshly obtained from gastric cancer patients or healthy volunteers. PBMCs were isolated by standard Ficoll-Hypaque density centrifugation (GE Healthcare) and stained with the following antibody mix: FITC-conjugated anti-human CD2, CD3, CD14, CD16, CD19, CD56, and CD235a (eBioscience, USA); Allophycocyanin- (APC-) conjugated anti-human ICOS and Peridinin-Chlorophyll Protein Complex- (PerCP-) conjugated anti-human IL-17RB (R & D, USA). The isotype control antibody was used in all cases. For FACS phenotype analysis, data were acquired on an Accuri C6 (BD company) and analyzed with FlowJo software (TreeStar, Inc.).

BM mononuclear cells (MNCs) were purified using Ficoll-Hypaque density centrifugation (GE Healthcare, Dornstadt, Germany). MNCs were incubated with CD34⁺ magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and separated using an AutoMACS Pro machine (Miltenyi Biotec) according to the manufacturer’s recommendations. This total (further unsorted) CD34⁺ cell population (referred to as CD34⁺ hematopoietic stem/progenitor cells, HSPCs) was used to prepare RNA. Total RNA was extracted using acid guanidine-thiocyanate-phenol–chloroform method and treated with DNase I (Qiagen, Germantown, MD, USA). RNA quality was assessed using 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA), and RNA integrity numbers were greater than 8 for all samples. cDNA libraries were constructed using NEBNext kit (New England Biolabs, Ipswich, MA, USA). Sequencing was performed on Illumina HiSeq4000 or NovaSeq (Illumina, San Diego, CA, USA).
The raw data have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) database under accession number PRJNA679200.