3. Twenty hours after target cell injection, spleens are harvested
from immunized mice, processed to single-cell suspensions and
acquired by flow cytometry to identify target cells and determine CTL killing (Fig. 2).
4. % Target cell killing in an individual mouse is determined
relative to the presence of nontarget cells in that same mouse
and is also compared to the average of all naı ¨ve mice, Target
cells to Nontarget cells (Fig. 3).
2 Materials
2.1 Vaccine
Formulation
and Immunization
1. Adjuvant: Sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp(1,4)-β-D-Glcp-(1,1)-archaeol)
archaeosomes
(National
Research Council Canada).
2. Antigen: Ovalbumin (Hyglos GmbH, Bernried am Starnberger
See, Germany).
3. Negative control: Phosphate-buffered saline (PBS) (pH 7.4).
4. Insulin syringes, 1.0 cc.
2.2 Spleen Collection
and Processing
1. H-2Kb OVA (257–264) peptide, SIINFEKL (JPT Peptide
Technologies, Berlin, Germany) (see Note 3).
2. Fetal bovine serum (FBS).
Fig. 1 Illustration of in vivo CTL cellular preparation workflow. Spleens are
harvested from donor C57BL/6 mice and processed into a single cell
suspension. Cells are divided into two equal portions, one group is identified
as “Target” cells and incubated with the CD8
+ T cell peptide epitope of the
ovalbumin protein, SIINFEKL; the other group of cells identified as “Nontarget”
cells is left untouched. Next, “Target” cells are stained with a high concentration
of CFSE and “Nontarget” cells are stained with a low concentration of CFSE.
“Target” and “Nontarget” cells are combined and injected intravenously into
experimental syngeneic C57BL/6 mice
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
551
from immunized mice, processed to single-cell suspensions and
acquired by flow cytometry to identify target cells and determine CTL killing (Fig. 2).
4. % Target cell killing in an individual mouse is determined
relative to the presence of nontarget cells in that same mouse
and is also compared to the average of all naı ¨ve mice, Target
cells to Nontarget cells (Fig. 3).
2 Materials
2.1 Vaccine
Formulation
and Immunization
1. Adjuvant: Sulfated lactosylarchaeol (SLA; 6
0 -sulfate-β-D-Galp(1,4)-β-D-Glcp-(1,1)-archaeol)
archaeosomes
(National
Research Council Canada).
2. Antigen: Ovalbumin (Hyglos GmbH, Bernried am Starnberger
See, Germany).
3. Negative control: Phosphate-buffered saline (PBS) (pH 7.4).
4. Insulin syringes, 1.0 cc.
2.2 Spleen Collection
and Processing
1. H-2Kb OVA (257–264) peptide, SIINFEKL (JPT Peptide
Technologies, Berlin, Germany) (see Note 3).
2. Fetal bovine serum (FBS).
Fig. 1 Illustration of in vivo CTL cellular preparation workflow. Spleens are
harvested from donor C57BL/6 mice and processed into a single cell
suspension. Cells are divided into two equal portions, one group is identified
as “Target” cells and incubated with the CD8
+ T cell peptide epitope of the
ovalbumin protein, SIINFEKL; the other group of cells identified as “Nontarget”
cells is left untouched. Next, “Target” cells are stained with a high concentration
of CFSE and “Nontarget” cells are stained with a low concentration of CFSE.
“Target” and “Nontarget” cells are combined and injected intravenously into
experimental syngeneic C57BL/6 mice
A Method to Evaluate In Vivo CD8
+ T Cell Cytotoxicity in a Murine Model
551