Author: Lamborn, Ian T.; Jing, Huie; Zhang, Yu; Drutman, Scott B.; Abbott, Jordan K.; Munir, Shirin; Bade, Sangeeta; Murdock, Heardley M.; Santos, Celia P.; Brock, Linda G.; Masutani, Evan; Fordjour, Emmanuel Y.; McElwee, Joshua J.; Hughes, Jason D.; Nichols, Dave P.; Belkadi, Aziz; Oler, Andrew J.; Happel, Corinne S.; Matthews, Helen F.; Abel, Laurent; Collins, Peter L.; Subbarao, Kanta; Gelfand, Erwin W.; Ciancanelli, Michael J.; Casanova, Jean-Laurent; Su, Helen C.
Title: Recurrent rhinovirus infections in a child with inherited MDA5 deficiency Document date: 2017_7_3
ID: vipx6t7e_55
Snippet: All lentivirus preparations were titered on 293T cells to determine the concentration of infectious units. 100,000 293T cells were resuspended in complete IMDM containing 8 µg/ml polybrene (Sigma-Aldrich) and 10 µl of diluted lentivirus to a final volume of 1 ml. The suspension was added to a 24-well plate (Corning) and spin-infected at 1,350× g for 30 min at 35°C. 48 h later, cell monolayers were washed twice with PBS, trypsinized (Gibco), t.....
Document: All lentivirus preparations were titered on 293T cells to determine the concentration of infectious units. 100,000 293T cells were resuspended in complete IMDM containing 8 µg/ml polybrene (Sigma-Aldrich) and 10 µl of diluted lentivirus to a final volume of 1 ml. The suspension was added to a 24-well plate (Corning) and spin-infected at 1,350× g for 30 min at 35°C. 48 h later, cell monolayers were washed twice with PBS, trypsinized (Gibco), transferred to 14 ml round bottom FACS tubes (Falcon), washed with PBS, and resuspended in PBS containing 0.5 µg/ml propidium iodide (Sigma-Aldrich). Single cell suspensions were analyzed on a BD FAC SCanto II to determine the percentages of GFP + cells among the propidium iodide low (live) populations. Lentivirus dilutions transducing between 2 and 15% of target cells were used to determine concentration of each preparation.
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