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Lentiviral transduction of U937 cells

AB Abhishek Bhattacherjee
JJ Jaesoo Jung
SZ Sameera Zia
MH Madelene Ho
GE Ghazaleh Eskandari-Sedighi
CL Chris D. St. Laurent
KM Kelli A. McCord
AB Arjun Bains
GS Gaurav Sidhu
SS Susmita Sarkar
JP Jason R. Plemel
MM Matthew S. Macauley
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Lentivirus was titered as previously described [17]. 250,000 U937 cells were plated in a 12 well plate in 500 μl growth media. Lentivirus was added to each well at a MOI = 1 and incubated in a tissue culture incubator. 16 h post-transduction, media was topped up to 2 ml. 72 h following transduction, 300 μg/ml zeocin was added to each well as a selection agent to kill untransduced cells. Cells were monitored daily for cell death, and every 2 days the media was changed and re-supplemented with 300 μg/ml zeocin. After 1 week, cells were assayed on a flow cytometer to assess the purity of the selected population via the fluorescent marker mAmetrine. Cells were found to be > 97% mAmetrine positive after 1 week of zeocin selection. Phagocytosis assays were done 3 days after removing zeocin from the media.

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