fraction of Target cells remaining in one mouse. See Fig. 3B for
an example calculation.
4 Notes
1. The number of mice used per group can vary depending on the
comparisons to be made in the experiment. We typically use ten
mice per group when comparing vaccine formulations and
repeat studies at least once. SLA is routinely used as an adjuvant
in our immunization studies. For testing other adjuvants, manufacturer’s recommendations should be followed. We routinely
immunize with 50 or 100 μl of vaccine formulation by intramuscular injection in the tibialis anterior or by subcutaneous
injection at the base of the tail, respectively.
2. A total of 20 million donor cells are needed for each experimental mouse. Depending on the age of the donor mouse,
spleen cell numbers will vary. The typical cell yield from one
mouse after processing is 40–60 million cells.
3. SIINFEKL peptide was chosen because it is the immunodominant CD8
+ T cell epitope for OVA protein in C57BL/6 mice.
If using a different protein or mouse strain, a CD8
+ T cell
peptide epitope must be chosen for that protein in that host.
4. It is important to use room temperature PBS as cold PBS can
negatively affect cell viability when staining with CFSE.
5. FACS buffer (also known as cell staining buffer) commonly
includes a protein carrier such as FBS or bovine serum albumin
(BSA) to reduce nonspecific binding of antibodies, but it also
provides a nutrient for cells in solution during prolonged tissue
processing. EDTA is also included to prevent cell clumping.
6. Isoflurane is routinely used for clinical anesthesia of mice in our
studies. Other anesthetizing agents may also be used depending on availability and approval by the institutional animal care
and use committee.
7. The frosted end of a glass slide is typically abrasive, similar to
sandpaper, and has enough grit to aid in the manual dissociation of the spleen. Not all frosted glass slides have the abrasive
frosted end and other methods of mechanical spleen cell dissociation are acceptable [8].
8. The volume specified here is to achieve a solution of welldispersed cells that can be accurately divided into two equal
portions.
9. Some peptides are difficult to dissolve in aqueous solutions;
therefore, we use the polar aprotic solvent DMSO to make all
peptide solutions. The final concentration of DMSO in the cell
556
Felicity C. Stark et al.
an example calculation.
4 Notes
1. The number of mice used per group can vary depending on the
comparisons to be made in the experiment. We typically use ten
mice per group when comparing vaccine formulations and
repeat studies at least once. SLA is routinely used as an adjuvant
in our immunization studies. For testing other adjuvants, manufacturer’s recommendations should be followed. We routinely
immunize with 50 or 100 μl of vaccine formulation by intramuscular injection in the tibialis anterior or by subcutaneous
injection at the base of the tail, respectively.
2. A total of 20 million donor cells are needed for each experimental mouse. Depending on the age of the donor mouse,
spleen cell numbers will vary. The typical cell yield from one
mouse after processing is 40–60 million cells.
3. SIINFEKL peptide was chosen because it is the immunodominant CD8
+ T cell epitope for OVA protein in C57BL/6 mice.
If using a different protein or mouse strain, a CD8
+ T cell
peptide epitope must be chosen for that protein in that host.
4. It is important to use room temperature PBS as cold PBS can
negatively affect cell viability when staining with CFSE.
5. FACS buffer (also known as cell staining buffer) commonly
includes a protein carrier such as FBS or bovine serum albumin
(BSA) to reduce nonspecific binding of antibodies, but it also
provides a nutrient for cells in solution during prolonged tissue
processing. EDTA is also included to prevent cell clumping.
6. Isoflurane is routinely used for clinical anesthesia of mice in our
studies. Other anesthetizing agents may also be used depending on availability and approval by the institutional animal care
and use committee.
7. The frosted end of a glass slide is typically abrasive, similar to
sandpaper, and has enough grit to aid in the manual dissociation of the spleen. Not all frosted glass slides have the abrasive
frosted end and other methods of mechanical spleen cell dissociation are acceptable [8].
8. The volume specified here is to achieve a solution of welldispersed cells that can be accurately divided into two equal
portions.
9. Some peptides are difficult to dissolve in aqueous solutions;
therefore, we use the polar aprotic solvent DMSO to make all
peptide solutions. The final concentration of DMSO in the cell
556
Felicity C. Stark et al.