With this in mind, our experiments were conducted with an extensive panel of dump antibodies as well as the two main published protocols for stringency in tetramer-based analysis: (i) dump tetramers (Newell et al., 2009; Savage et al., 1999), or (ii) two-color tetramers (Nelson et al., 2015; Obar et al., 2008; Stetson et al., 2002).
It is, however, conceivable that HCV-specific CD4+ T cells in contrast to CD8+ T cells are lost in vitro during the expansion step and further studies with an optimised protocol for direct ex-vivo HLA class II tetramer staining of liver infiltrating lymphocytes will need to be performed to resolve this issue.
For tetramer staining, PBMCs were stained with PE labeled AMQMLKETI tetramer (NIH Tetramer Core Facility), APC-CD3, and Pacific Blue-CD8a.
For tetramer staining, DbGP33 (MHC class I) and I-AbGP61 (MHC class II) tetramers were used at a 1∶50 1∶100 dilution in FACS buffer.
In summary, we have developed a rapid "one-pot, mix-and-read" strategy for tetramer production.
For tetramer, the purified DR0401 was biotinylated according to manufacturer's instruction (Avidity, Aurora, CO).
T cells from each donor were double labeled for tetramer and IC-IFNγ (Figure 9).
This implies that D9's TCR avidity for tetramer is lower than its cross-reactive counterparts.
For tetramer analysis, cells were stained with Db Smcy PE-conjugated tetramer as described 45.
It is not required for tetramer formation, 40) although it does contribute to stabilization of the tetramers.
