Nucleofection of CD8 T cells were performed following the manufacturer’s (Lonza) protocol, and as described in detail elsewhere (3 (link)). In short, 1×106 CD8 T cells were resuspended in 100uL of Nucleofector buffer (Lonza) along with the RNP complex, with or without the p53siRNA (Santa Cruz, sc-29436) in a glass cuvette (Lonza). Nucleofector 2b device (Lonza) with Nucleofector program X001 was used. Following nucleofection, cell suspension was removed from cuvette, placed in Eppendorf tube with 100 uL of RPMI 1640 (Gibco) containing 10% fetal calf serum (Gibco). After incubation at 37°C for 10 min, the CD8 T cells were then placed in culture containing the T cell growth media (Lonza) or transferred intravenously into recipient mouse.
Protocol detail
Nucleofection of CD8 T Cells
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Buffer
Cd8 t cells
Cell
Device
Fetal calf serum
Growth media
Mouse
T cell
Corresponding Organization :
Other organizations : University of Georgia, University of Iowa
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Variable analysis
independent variables
- Presence or absence of p53siRNA (Santa Cruz, sc-29436)
- Not explicitly mentioned
- Number of CD8 T cells (1×10^6)
- Nucleofector buffer (Lonza)
- Nucleofector 2b device (Lonza) with Nucleofector program X001
- Incubation time (10 min) after nucleofection
- T cell growth media (Lonza)
- Positive control: CD8 T cells nucleofected with RNP complex
- Negative control: CD8 T cells nucleofected with RNP complex and p53siRNA
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