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.
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.