3.1 Vaccine
Preparation
3.2 Anesthesia
and Intramuscular
Injection
1. Turn on the oxygen flowmeter of the isoflurane anesthesia
machine and set the rate at 2 L/min (see Note 6).
2. Turn on the isoflurane vaporizer and set to 3% [6].
3. After 5 min, place the mouse in the anesthetizing chamber.
4. Once the mouse is completely anesthetized, disinfect the injection site above the TA muscle by spraying with 70% ethanol.
5. Draw 50 μL of antigen, vaccine or negative control using a
0.5 cc insulin syringe.
6. Insert the syringe needle into the polyethylene (PE)-20 tubing,
and cut the tubing so that around 2 mm of the needle bevel is
exposed when the other end of the tubing is flush against the
plastic hub (this will help keep the injection depth consistent).
7. Slowly inject total volume into the TA muscle (see Note 7).
8. Transfer the mouse back into cage after injection to allow full
recovery from anesthesia.
9. Turn off the isoflurane vaporizer and keep the oxygen on for
5 min to remove residual isoflurane in the anesthetizing
chamber.
10. Turn off the oxygen flowmeter of the isoflurane machine.
3.3 Muscle
Collection
and Processing
1. Anesthetize the mouse using the isoflurane anesthesia machine
as described previously.
2. Perform cervical dislocation once the mouse is completely
anesthetized (see Note 8).
3. Carefully isolate the TA muscle using surgical scissors and
forceps at day 1 or 3 postinjection. Keep TA muscle in 1 mL
R10 complete medium on ice or at 4
C until ready for tissue
processing.
4. Thaw a frozen aliquot of 0.2% Collagenase Type IV digestion
buffer on ice or prepare fresh (see Note 9). Transfer 2 mL of the
digestion buffer onto a petri dish. Alternatively, keep the digestion buffer on ice until ready for tissue processing.
5. Tilt the petri dish until the 0.2% Collagenase Type IV digestion
buffer settles onto the bottom end. Place the TA muscle in the
digestion buffer and mince to approximately 1–2 mm in length
using surgical scissors. Ensure that the TA muscle remains in
the digestion buffer while mincing to prevent drying of the
tissue.
6. Transfer the 0.2% Collagenase Type IV digestion buffer containing the minced TA muscle into a 15-mL Falcon™ conical
Cell Recruitment and Antigen Uptake and Distribution
517
Preparation
3.2 Anesthesia
and Intramuscular
Injection
1. Turn on the oxygen flowmeter of the isoflurane anesthesia
machine and set the rate at 2 L/min (see Note 6).
2. Turn on the isoflurane vaporizer and set to 3% [6].
3. After 5 min, place the mouse in the anesthetizing chamber.
4. Once the mouse is completely anesthetized, disinfect the injection site above the TA muscle by spraying with 70% ethanol.
5. Draw 50 μL of antigen, vaccine or negative control using a
0.5 cc insulin syringe.
6. Insert the syringe needle into the polyethylene (PE)-20 tubing,
and cut the tubing so that around 2 mm of the needle bevel is
exposed when the other end of the tubing is flush against the
plastic hub (this will help keep the injection depth consistent).
7. Slowly inject total volume into the TA muscle (see Note 7).
8. Transfer the mouse back into cage after injection to allow full
recovery from anesthesia.
9. Turn off the isoflurane vaporizer and keep the oxygen on for
5 min to remove residual isoflurane in the anesthetizing
chamber.
10. Turn off the oxygen flowmeter of the isoflurane machine.
3.3 Muscle
Collection
and Processing
1. Anesthetize the mouse using the isoflurane anesthesia machine
as described previously.
2. Perform cervical dislocation once the mouse is completely
anesthetized (see Note 8).
3. Carefully isolate the TA muscle using surgical scissors and
forceps at day 1 or 3 postinjection. Keep TA muscle in 1 mL
R10 complete medium on ice or at 4
C until ready for tissue
processing.
4. Thaw a frozen aliquot of 0.2% Collagenase Type IV digestion
buffer on ice or prepare fresh (see Note 9). Transfer 2 mL of the
digestion buffer onto a petri dish. Alternatively, keep the digestion buffer on ice until ready for tissue processing.
5. Tilt the petri dish until the 0.2% Collagenase Type IV digestion
buffer settles onto the bottom end. Place the TA muscle in the
digestion buffer and mince to approximately 1–2 mm in length
using surgical scissors. Ensure that the TA muscle remains in
the digestion buffer while mincing to prevent drying of the
tissue.
6. Transfer the 0.2% Collagenase Type IV digestion buffer containing the minced TA muscle into a 15-mL Falcon™ conical
Cell Recruitment and Antigen Uptake and Distribution
517