5. Vaccine preparation methods will vary depending on the antigen and adjuvant used. The recommended maximum injection
volume of 50 μL is routinely used in our immunization
studies [7].
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 typical sites of intramuscular injection in mice are the TA,
gastrocnemius, and quadriceps leg muscle groups [8]. The TA
muscle is routinely used in our immunization studies to evaluate local and systemic responses, particularly immune cell
recruitment and the cellular uptake and distribution of antigen.
Intramuscular injection should only be performed by trained
personnel. Due to the small size of the TA muscle, extra care
must be taken when performing intramuscular injection to
minimize the risk of damage to surrounding tissue [7]. Practice
injection technique using tracker dyes, such as India ink and 1%
methylene blue [8, 9].
8. Mouse euthanasia through cervical dislocation should only be
performed under anesthesia by trained personnel.
9. Avoid repeated freeze–thaw cycles to prevent degradation of
Collagenase Type IV enzymatic activity.
10. Samples can be diluted further at 1:5 or 1:10 ratio with R10
complete medium in a fresh microcentrifuge tube prior to
addition of dye if cell concentration is too high. Furthermore,
cell counting can be performed using trypan blue and a hemocytometer if an automated cell counter is not readily available.
11. Label a tube for each fluorochrome-conjugated antibody used
for staining and add one drop of UltraComp eBeads™, as
recommended by the manufacturer. Add the appropriate
fluorochrome-conjugated antibody (0.03–1.0 μg; quantity
based on amount used to stain cells) to each tube and mix
thoroughly. Incubate for 15–30 min at 4
C in the dark.
Perform the same wash steps as the stained cells and resuspend
in 500 μL staining buffer.
12. Prepare negative and positive controls for LIVE/DEAD™
Fixable Dead Cell Stain. While the manufacturerrecommended working concentration of 1:1000 is a good
starting point, increasing it to 1:100 provides a strong positive
signal and allows clear distinction between live and dead cell
populations in this setting. Furthermore, spleen cells can be
used for the controls to avoid unnecessary usage of muscle and
LN samples. Collect and process the spleen in the same manner
as LNs. Bring the final volume of the single-cell suspension to
10 mL using R10 complete medium prior to counting the cells
Cell Recruitment and Antigen Uptake and Distribution
521
volume of 50 μL is routinely used in our immunization
studies [7].
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 typical sites of intramuscular injection in mice are the TA,
gastrocnemius, and quadriceps leg muscle groups [8]. The TA
muscle is routinely used in our immunization studies to evaluate local and systemic responses, particularly immune cell
recruitment and the cellular uptake and distribution of antigen.
Intramuscular injection should only be performed by trained
personnel. Due to the small size of the TA muscle, extra care
must be taken when performing intramuscular injection to
minimize the risk of damage to surrounding tissue [7]. Practice
injection technique using tracker dyes, such as India ink and 1%
methylene blue [8, 9].
8. Mouse euthanasia through cervical dislocation should only be
performed under anesthesia by trained personnel.
9. Avoid repeated freeze–thaw cycles to prevent degradation of
Collagenase Type IV enzymatic activity.
10. Samples can be diluted further at 1:5 or 1:10 ratio with R10
complete medium in a fresh microcentrifuge tube prior to
addition of dye if cell concentration is too high. Furthermore,
cell counting can be performed using trypan blue and a hemocytometer if an automated cell counter is not readily available.
11. Label a tube for each fluorochrome-conjugated antibody used
for staining and add one drop of UltraComp eBeads™, as
recommended by the manufacturer. Add the appropriate
fluorochrome-conjugated antibody (0.03–1.0 μg; quantity
based on amount used to stain cells) to each tube and mix
thoroughly. Incubate for 15–30 min at 4
C in the dark.
Perform the same wash steps as the stained cells and resuspend
in 500 μL staining buffer.
12. Prepare negative and positive controls for LIVE/DEAD™
Fixable Dead Cell Stain. While the manufacturerrecommended working concentration of 1:1000 is a good
starting point, increasing it to 1:100 provides a strong positive
signal and allows clear distinction between live and dead cell
populations in this setting. Furthermore, spleen cells can be
used for the controls to avoid unnecessary usage of muscle and
LN samples. Collect and process the spleen in the same manner
as LNs. Bring the final volume of the single-cell suspension to
10 mL using R10 complete medium prior to counting the cells
Cell Recruitment and Antigen Uptake and Distribution
521