Its solution

MV4–11, THP-1, and U937 cell lines were purchased from the American Type Culture Collection (2006, 2014, and 2002, respectively; Manassas, VA). OCI-AML3 cell line was purchased from the German Collection of Microorganisms and Cell Cultures (2011; DSMZ, Braunschweig, Germany). The CMS cell line was a gift from Dr. A Fuse from the National Institute of Infectious Diseases, Tokyo, Japan (2004). The cell lines have not been authenticated since receiving them in our laboratory. The cell lines were cultured in RPMI 1640 (or αMEM for OCI-AML3 cells) media with 10–20% fetal bovine serum (Life Technologies, Carlsbad, CA) and 2 mM L-glutamine, plus 100 U/ml penicillin and 100 µg/ml streptomycin, in a 37 °C humidified atmosphere containing 5% CO₂/95% air. Cell lines were tested for the presence of mycoplasma.
Diagnostic AML blast samples derived from patients were purified by standard Ficoll-Hypaque density centrifugation, then cultured in RPMI 1640 with 20% fetal bovine serum, ITS solution (Sigma-Aldrich, St. Louis, MO) and 20% supernatant of the 5637 bladder cancer cell line (as a source of granulocyte-macrophage colony-stimulating factor, granulocyte colony-stimulating factor, interleukin-1 beta, macrophage colony-stimulating factor, and stem cell factor (12 (link), 16 (link), 17 (link))).

Eyecups with intact RPE monolayers were prepared as previously described [1 (link),3 (link),4 (link)]. In brief, eyes from euthanized healthy C57BL/6J mice were enucleated, placed in ice cold DMEM (Lonza, Walkersvile, PA), where external muscles, and connective tissue were cut from the eyes. A circumferential incision was performed below the level of the ciliary body, and the anterior segment and lens was discarded. The neural retina was gently lifted off the RPE by microsurgical forceps. Each RPE eyecup were placed into individual wells of a 96-well round-bottom culture plate submerged in 200 μL of serum-free media consisting of RPMI 1640, 0.1 M HEPES, NEAA, L- glutamine, sodium pyruvate, 0.1% bovine serum albumin, 1% gentamicin, and supplemented with 0.1 x ITS+ solution (Sigma Chemical, St. Louis, MO). The conditioned media (CM) from the eyecup cultures were removed 48 hours after incubation, and used in the assays.

Diagnostic blast samples were obtained from the First Hospital of Jilin University, Changchun, China. Written informed consent was provided according to the Declaration of Helsinki. This study was approved by the Human Ethics Committee of The First Hospital of Jilin University. The samples were purified by standard Ficoll-Hypaque density centrifugation, then cultured in RPMI 1640 with 20% fetal bovine serum, ITS solution (Sigma-Aldrich) and 20% supernatant of the 5637 bladder cancer cell line (as a source of granulocyte-macrophage colony-stimulating factor).^{48 (link)}