Fix perm and perm wash solution

To detect VP11/12-specific cytolytic CD8⁺ T cells in PBMC, spleen, TG and cornea cells, intracellular CD107a/b cytotoxicity assay was performed as described by Betts et al. (2003) with a few modifications (37 (link), 38 (link)). Briefly, 1×10⁶ PBMCs from patients, in addition to spleen cells, DLN cells and TG cells from HSV-infected mice infected, were transferred into 96-well V-bottomed FACS plates (BD) in R10 medium and stimulated with 10 different VP11/12 peptides (10 μg/mL) in the presence of anti-CD107a-FITC and CD107b-FITC (BD PHArmingen) and BD-Golgi stop (10μg /ml) for 5–6 hours at 37°C. PHA (10 μg/mL) (Sigma) and no peptide were used as positive and negative controls, respectively. At the end of the incubation period, the cells were harvested into separate tubes and washed once with FACS buffer and then stained with PE-conjugated anti-human CD8 antibody for 30 minutes. Cells were then fixed, permeabilized and stained with additional antibodies against IFN-γ and TNF- α using FIX/PERM and PERM/Wash solution (BD).

To detect VP11/12-specific cytolytic CD8⁺ T cells in PBMC and cornea cells, intracellular CD107a/b cytotoxicity assay was performed as described by Betts et al. with a few modifications (25 (link), 26 (link)). Briefly, 1×10⁶ PBMCs from patients, in addition to spleen cells, DLN cells and TG cells from mice infected with HSV, were transferred into 96-well V bottom FACS plates (BD) in R10 medium and stimulated with 10 different VP11/12 peptides (10μg/mL) in the presence of anti-CD107a-FITC and CD107b-FITC (BD Pharmingen) and BD-Golgi stop (10ug/ml) for 5–6 hours at 37°C. ConA (10μg/mL) (Sigma) and no peptide were used as positive and negative controls, respectively. At the end of the incubation period the cells were harvested into separate tubes and washed once with FACS buffer and then stained with PE-conjugated anti-human CD8 antibody for 30 min. Cells were fixed, permeabilized, and stained with additional antibodies against IFN-γ using FIX/PERM and PERM/Wash solution (BD).