Author: Unknown. Date: 12/5/95
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 also 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
See also: General Tissue Culture S.O.P. in this manual.
Always wear gloves and lab coat when handling animals or animal carcasses. Wear a face mask if youÕre allergic to mice or to prevent the onset of allergies.
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).
If TG broth is accidently injected into hand, wash wound immediately with lots of water. Report this to lab safety officer.
Needles must be disposed of in a biohazard sharps container.
Animal carcasses must be frozen and disposed of by Research Animal Resources. There is a freezer on the 19th floor of Moos Tower.
1. 4-6 days before harvesting, inject 1.0ml Thiglycollate* broth into mice. (This elicits activated macrophages to the peritoneum.)
2. To harvest the cells, prepare the following:
2 sterile scissors
1 sterile forceps
a 60 cc syringe filled with buffer( HBSS + 1% FCS) with a 23 g needle
sterile 50 ml tubes
10 cc syringe(s) with 19g needle(s)
3. Euthanize the animal.
4. Wet with alcohol, snip skin over abdomen and pull back completely to reveal the peritoneum, being careful not to puncture the peritoneal wall.
5. Inject 8-10 cc ice cold buffer (using the 23g needle) through the fat pads. (This ÒplugsÓ the hole made by the needle, so the buffer doesnÕt flow back out.) Agitate the carcass gently.
6. Insert the 19g needle (with dry 10 cc syringe attached) and extract as much liquid as possible. Note: be very careful not to puncture an intestine. Discard the cells if a puncture occurs.
7. *optional: snip the peritoneal wall at the top. Insert a sterile pastuer pipette and try to get remaining fluid while holding the peritoneal wall up with forceps.
8. Pool the cells from all mice. Wash 3 times with cold buffer.
9. Dilute cells to 1x106 /ml in RP10. Incubate for 1-2 hours at 37oC to adhere. Wash gently but thoroughly.
yield = approx. 1x107 cells per mouse.
*Thioglycollate Broth:
10.1 g Brewers thioglycollate (DIFCO)
add 250 ml distilled water
Boil for 1 minute to dissolve
autoclave to sterilize