To analyze the oligomerization process, we measured the arc-length distributions of wild-type suilysin in the pore state and of the disulphide-locked mutant in prepore (−DTT) and reduced, pore (+DTT) configurations, based on negative stain EM analysis under the same conditions.
The experimental data here are based on negative-stain EM images on monolayers of egg PC:cholesterol (45:55%), incubated at 37°C.
Gating was implemented based on negative control staining profiles.
Quadrants were determined based on negative control staining with a corresponding isotype antibody.
Three HGA samples from our initial dataset were thus excluded based on negative INI1 staining.
Our results demonstrate that Eiger-mediated death in R cells is caused by apoptosis, based on negative PI staining (Supplementary Figure S3B) and mutants of necroptosis components, including loss-of-function (LOF) PGMAM5 and Drp1 (Supplementary Figure S3C).
Non-calcified aortic valves were identified based on negative routine von Kossa staining.
The procedure is based on negative magnetic separation and results in highly purified, functional cell populations (purity > 90% as confirmed with Wright-Giemsa stain) (Kulkarni et al., 2011).
Viable cells were gated based on their negative staining with the Live/Dead® dye (Invitrogen), and these cells were finally analyzed for anti-human CD8 antibodies and HLA-tetramer stainings.
