Human elmo1 targeting or control shrna pgfp 5 rs expression vectors

HL-60 cell line (ATCC) was grown in IMDM containing 20% FBS (Gemini Bio-Products) and 1% penicillin/streptomycin/glutamine (Gemini Bio-Products) and routinely tested negative for Mycoplasma contamination. For differentiation into neutrophil-like cells, HL-60 cells were treated with 1.25% DMSO (Sigma) for 4 days⁵⁴ and then cultured without DMSO for 16h. For generating Elmo1 knockdown cells, HL-60 cells were transfected with 10 μg of human Elmo1 targeting or control shRNA pGFP-V-RS expression vectors (OriGene Technologies, Inc.) using electroporation using a 250 V, 25 ms pulse. After 24h, stably transfected cells were selected with 0.2 μg/ml puromycin (Gibco) and single cell cloning was performed by serial dilution. A total of three control and four Elmo1 shRNA clones were analyzed. Differentiated cells (1 × 10⁵ cells per well) were seeded in top chambers of 3 μm polycarbonate Transwell plates in 100 μl of HBSS containing 1% bovine serum albumin, as described for neutrophils. Bottom chambers contained 600 μl of 100 nM leukotriene B4. After 1 hour at 37°C, the cells in the bottom wells were collected and subjected to flow cytometry analysis, as described above. The data is presented as percent of input cells.