Protocol detail

Quantifying Mart1-specific CD8+ T Cells

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After TAME, 1500 naïve and memory Mart1-specific CD8⁺ T cells were sorted into RLT Buffer (Qiagen, France). Total RNA was extracted (Qiagen Microkit). cDNA were generated using the Supercript II enzyme (Invitrogen, France). RT-PCR reactions, thermal cycling conditions, calculations for relative usage of each Vβ family, and immunoscope profiles were performed as previously described (Alanio et al., 2013 (link); Bouvier et al., 2011 (link)) (Supplementary file 2).

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Alanio C., Nicoli F., Sultanik P., Flecken T., Perot B., Duffy D., Bianchi E., Lim A., Clave E., van Buuren M.M., Schnuriger A., Johnsson K., Boussier J., Garbarg-Chenon A., Bousquet L., Mottez E., Schumacher T.N., Toubert A., Appay V., Heshmati F., Thimme R., Pol S., Mallet V, & Albert M.L. (2015). Bystander hyperactivation of preimmune CD8+ T cells in chronic HCV patients. eLife, 4, e07916.

Publication 2015

RLT Buffer Microkit

Buffer Cdna Enzyme Mart1 Memory t cells Rt pcr Tame

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Variable analysis

dependent variables

Number of naïve and memory Mart1-specific CD8+ T cells

control variables

Thermal cycling conditions
RT-PCR reactions
Calculations for relative usage of each Vβ family
Immunoscope profiles

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

Annotations

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