Journal: Nature protocols

Article Title: Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function

doi: 10.1038/nprot.2017.083

Figure Lengend Snippet: Reduced cell viability after flow-cytometric sorting leads to instability of transferred T cells in vivo. Wild-type P14 cells that were activated in vitro were transduced with empty-GFP RV on day 1, as depicted in Figure 1. The following day (day 2 after in vitro stimulation), GFP+ P14 cells were enriched by flow-cytometric sorting using the BD ARIA II with a 100-μm nozzle and 20-p.s.i. condition. After sorting, viable cell numbers were carefully determined by 7-AAD and trypan blue staining. As a control, P14 cells without sorting were used, and 1.5 × 105 live cells of each cell type were adoptively transferred into LCMV-Arm-infected recipients. On day 8, expansion of P14 cells was determined in blood. (a) Flow plots showing physical cell size and purity of GFP+ CD8+ cells before and after flow sorting on day 2. Purity of GFP+ CD8+ cells was over 95%. (b) 7-AAD histograms gated on ‘live’ or ‘dying’ cells based on forward scatter (FS) versus side scatter (SS) plot in a, showing that the majority of small cells after sorting are dying. (c) Cell recovery (%) based on the number of total live (trypan blue negative) cells in sorting. (d) Flow plots showing frequencies of P14 cells among total CD8+ cell gate in blood at 8 d.p.i. (e) Graph showing the number of P14 cells per 1 × 106 cells in blood at 8 d.p.i. *P< 0.0005 (two-tailed t test). Data are representative of two experiments (n = 5–15 per group). All animal experiments depicted in this figure were performed in accordance with the institutional animal care and use guidelines of the University of Pennsylvania.

Article Snippet: 00413612 and 10053295) Easystep Magnet (15 ml; StemCell, cat. no. 18001) Vortex mixer (Scientific Industries, cat. no. SI-0236) Microcentrifuge (Eppendorf, cat. no. 5424) Centrifuge (Eppendorf, cat. no. 5810R, 15-Amp version) Rotor for swing buckets (Eppendorf, cat. no. A-4-81) MTP/Flex bucket for Rotor A-4-81 (Eppendorf, cat. no. 022638866) 500-ml rectangular bucket, for Rotor A-4-81 (Eppendorf, cat. no. 022638629) Adaptor for five conical 50-ml tubes (Eppendorf, cat. no. 022638769) Adaptor for 12 conical 15-ml tubes (Eppendorf, cat. no. 022638742) Flow cytometric analyzer (BD Biosciences, model no. LSR II. running DIVA 8.0) Flow cytometric sorter (BD Biosciences, model no.

Techniques: In Vivo, In Vitro, Transduction, Staining, Infection, Two Tailed Test

Journal: Nature protocols

Article Title: Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function

doi: 10.1038/nprot.2017.083

Figure Lengend Snippet: Reduced cell viability after flow-cytometric sorting leads to instability of transferred T cells in vivo. Wild-type P14 cells that were activated in vitro were transduced with empty-GFP RV on day 1, as depicted in Figure 1. The following day (day 2 after in vitro stimulation), GFP+ P14 cells were enriched by flow-cytometric sorting using the BD ARIA II with a 100-μm nozzle and 20-p.s.i. condition. After sorting, viable cell numbers were carefully determined by 7-AAD and trypan blue staining. As a control, P14 cells without sorting were used, and 1.5 × 105 live cells of each cell type were adoptively transferred into LCMV-Arm-infected recipients. On day 8, expansion of P14 cells was determined in blood. (a) Flow plots showing physical cell size and purity of GFP+ CD8+ cells before and after flow sorting on day 2. Purity of GFP+ CD8+ cells was over 95%. (b) 7-AAD histograms gated on ‘live’ or ‘dying’ cells based on forward scatter (FS) versus side scatter (SS) plot in a, showing that the majority of small cells after sorting are dying. (c) Cell recovery (%) based on the number of total live (trypan blue negative) cells in sorting. (d) Flow plots showing frequencies of P14 cells among total CD8+ cell gate in blood at 8 d.p.i. (e) Graph showing the number of P14 cells per 1 × 106 cells in blood at 8 d.p.i. *P< 0.0005 (two-tailed t test). Data are representative of two experiments (n = 5–15 per group). All animal experiments depicted in this figure were performed in accordance with the institutional animal care and use guidelines of the University of Pennsylvania.

Article Snippet: 00413612 and 10053295) Easystep Magnet (15 ml; StemCell, cat. no. 18001) Vortex mixer (Scientific Industries, cat. no. SI-0236) Microcentrifuge (Eppendorf, cat. no. 5424) Centrifuge (Eppendorf, cat. no. 5810R, 15-Amp version) Rotor for swing buckets (Eppendorf, cat. no. A-4-81) MTP/Flex bucket for Rotor A-4-81 (Eppendorf, cat. no. 022638866) 500-ml rectangular bucket, for Rotor A-4-81 (Eppendorf, cat. no. 022638629) Adaptor for five conical 50-ml tubes (Eppendorf, cat. no. 022638769) Adaptor for 12 conical 15-ml tubes (Eppendorf, cat. no. 022638742) Flow cytometric analyzer (BD Biosciences, model no. LSR II. running DIVA 8.0) Flow cytometric sorter (BD Biosciences, model no.

Techniques: In Vivo, In Vitro, Transduction, Staining, Infection, Two Tailed Test