Fast green neutral red

CD34⁺ cells were transduced with MISSION lentiviral shRNA
transduction particles (Sigma- Aldrich, St. Louis, MO) at an MOI of 5 for 20
hours. Cells were collected, the viruses washed away, and the media supplemented
with 100 nM IL-3 and IL-5 (R&D Systems, Minneapolis, MN) for the remainder
of the experiment to promote differentiation of the eosinophil lineage. Cells
were maintained at a concentration of 0.3–1.0 × 10⁶cell/mL for 14 or 21 days. Six days after the virus particles were removed, 2
μg/mL Puromycin (Sigma- Aldrich, St. Louis, MO) was added to the media in
order to select for cells transduced with the shRNA lentiviral particles. Cells
were stained using Fast Green/ Neutral Red and May-Grünwald Giemsa
(Sigma- Aldrich, St. Louis, MO) according to the manufacturer’s protocol.
Cell morphology on cytocentrifuge slides (Shandon Cytospin II, Thermo Fisher
Scientific, Waltham, MA) was evaluated by differential counts on sequential 40X
high power fields, counting at least 200 cells/slide.

Human CD34+ cells isolated by positive selection from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood of two healthy donors (provided by Dr. Harry Malech, NIAID/NIH) were cultured in IMDM (Invitrogen) supplemented with 10% FBS and GM-CSF (1 ng/ml), SCF, Flt3-L and TPO (each at 10 ng/ml), IL-5 (25 ng/ml), IL-3 (20 ng/ml) for 3 days, followed by IL-5 (25 ng/ml) only for another 21 days. Cells were collected on days 4, 6, 8, 10, 12, 15, 18, 21, and 24 of culture, and total RNA and cell lysates prepared at each time point. Eosinophil differentiation was assessed using Diff-Quik (Siemens Healthcare Diagnostics) and Fast Green/Neutral Red (Sigma-Aldrich) staining. At 21 days of culture, >60% of the cells were eosinophil-like by morphologic criteria and >20% were mature eosinophils based on Fast Green staining of cytoplasmic granules. Growth and viability were determined by light microscopic evaluation with trypan blue exclusion. Viability remained >95% throughout the 24-day experimental period.