Standard Operating Procedure:
Intrathymic injection of FITC/biotin
AUTHOR: Kris Hogquist
Date last revised: 3/24/09
IACUC
protocol # 0809A47801
Current Users of Procedure: Mike Weinreich,
Yan Xing
Past users: Tom McCaughtry, Troy Baldwin
Hazards:
Ketamine
and Buprenorphine are controlled substances and anyone
working with these needs to take the training session and be registered the
SBOP (State Board of Pharmacy). Go
to this website to register for the training https://onestop2.umn.edu/training/courseDetail.jsp?course=RA0001
FITC is an
irritant, Avoid contact with eyes, skin and clothing.
BIOTIN-NHS: Toxilogical data are not available. Handle as potentially harmful.
EXPOSURE TO
ANIMALS: Personnel involved with animals may be exposed to bites, scratches,
infectious agents, and allergens.
TETANOUS: a rare but potentially fatal bacterial infection is possible after any animal bite. See the Boynton Health Service brochures on animal bites and allergies in this manual.
CARBON DIOXIDE:
toxic gas, avoid inhaling.
SHARPS: take
appropriate cautions when working with needles
Protection:
Wear gloves and
lab coat.
Wear a face mask
if youÕre allergic to mice or to prevent the onset of allergies.
A hair net is
also recommended.
Have current
tetanus immunization.
If biten by a
mouse, immediately wash wound with soap and water. Seek medical assistance from
Boynton Health Service, if necessary (625-7900).
Waste:
Animal carcasses
must be frozen and disposed of by Research Animal Resources. There is a freezer
on the 2nd floor of Nils Hasselmo Hall.
Needles must be
disposed of in a biohazard sharps container.
Spill
clean-up: Isolate
the area. Use appropriate tools to put materials into a convenient waste
container.
Procedure:
Prepare reagents:
-Ketamine xylazine mix: load
120ul/mouse into a 1cc syringe
-Buprenorphine: dilute stock
1:10 in PBS, load 30ul/mouse into a 0.3 cc syringe
-FITC: thaw and spin, load
into a 100ul Hamilton syringe with 29 or 31 needle
-Eye moisture salve, 70%
EtOH, instruments (2 fine curved forceps, 1 fine scissors, 1 small scissors,
cotton swabs), stapler, immobilization apparatus
1. Anesthesia: Inject animals with 80ul ketamine/xylazine mix i.p. (use 1 cc tuberculin syringe, with 28g needle)
Note: as this anesthetic is a controlled substance, the
lab stock is kept in a locked box under JieÕs bench. Code is available to those who have completed controlled
substance traingin. From
commercial stocks of 100 mg/ml Ketmine and 100 mg/ml xylazine, technicians made
stocks that are 18 mg/ml ketamine and 1.8 mg/ml xylazine (10ml ketamine + 1ml
xylazine into 44ml PBS). These are
in 15 ml conical tubes. The
precise amount used should be recorded in the Disposition Log in the Controlled
substances notebook above JieÕs desk.
We
are approved to inject 100ul of this i.p., which is 90 mg/kg ketamine for the average mouse (25 g). RAR
recommends 25-75 mg/kg, so use 80ul to start. Anesthetic depth can be assessed
prior to the procedure by pinching the toe. If after 5 minutes, the toe reflex is still active,
the animal is not completely under, and a small amount more can be injected.
2. Surgery:
Apply
eye moisture salve to the eyes of each animal to prevent them from drying out
during the anesthesia period.
Place into an immobilization apparatus made with
rubber bands (see diagram, left).
Swab
the chest area with 70% EtOH.
Starting at the base of the sternum, make an incision through the skin,
straight up to about the base of the chin. Press skin away from centerline using cotton tipped swab.
Using
a clean fine scissors, start at the sternal notch, and make a 3-5 mm incision
down through the sternum, exactly at the centerline. Using two curved forceps, gently open the ribcage, removing
any tissue that may be directly above the thymus. The tip of the thymus should be visible. If not, you may need to make the
incision longer.
Inject
10ul of a solution of 1-3 mg/ml FITC in PBS. (This will not be completely in solution, so centrifuge 30
sec at max speed in a microfuge prior to administration.) Use a .3 cc tuberculin syringe, with a
29 g needle, or a 100ul hamilton syringe with a 29-31 g needle. If using a tuberculin syringe, it is
easy to inject too much, so load only the 10ul.
Using
a curved forceps hold the two flaps of skin together and apply 2-3
staples. Inject the analgesic
buprenorphine (Buprenex). Dilute a
.3mg/ml stock 1:10 in PBS, inject 30 ul of this (900ug) i.p.. Lay
the animal on its side in a clean cage on a heating pad sent on low to recover
(may take up to 2 hours for complete recovery).
3. Analysis
Thymic
emigration can be assessed up to 48 hours after dye injection.
Harvest thymus, spleen and
lymph nodes. Stain for CD4, CD8,
and any other markers needed. Keep
cells ice cold, and shielded from light as much as possible. Because the number of thymic emigrants
is low, stain 2 wells with 2x10e6 cells each, then pool prior to analysis. Be sure to analyze a control animal
that did not receive an injection of dye to establish instrument background
noise level. Collect at
least 500,000 live events from peripheral tissues.
4. Recovery
Observe the animals for
adverse health effects twice daily for three days post procedure. Insure that
the animals are active, eating, and drinking, and that the wound is healed. It
will be assumed that all animals are healthy post-surgery unless indicated
otherwise in the surgeon's notebook.
NOTE: surgeonÕs notebook indicating animal numbers and
surgery/post-surgery evaluation dates must be available to inspectors.
Include the following
ÒchecklistÓ in your notebook:
Anesthesia depth checked via toe-pinch
Ophthalmic ointment used
time of
surgery ______
time of
post-op assessment
1st
day _____
1st
day _____
2nd
day______
2nd
day_____
3rd
day_____
3rd
day_____