Author: KAH. 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.
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).
Animal carcasses must be frozen and disposed of by Research Animal Resources. There is a freezer on the 19th floor of Moos Tower.
Timed matings:
I time mate mice by placing 3 females with one male at the end of the day, and checking for plugs the next morning before 10 a.m. The day of the plug is considered day 1 (although technically it could be anywhere from 0 to 0.5 days).
Preparing the culture dishes:
1. Boil Millipore filters (#HAWG 013 00 ) 3 times in dH20. The final time cover with foil and when cool transfer into the hood. Place the filters in media or RPMI.
2. Prepare media. I use RP10: RMPI + 10 % h.i.FCS, + P/S/G + L-glutamine. FTOC can be done in serum free media as well. For this I use RPMI + P/S/G + L-glu + NEAA + Na pyruvate + 1% Nutridoma SP (BMB). If you're adding antibodies or drugs or peptide, include them now. Place 2 ml of media in each well of a Costar 6 well plate.
3. Sterily remove a strip of gel-foam (NDC 0009-0315-03, Upjohn) and cut it into three squares, placing each square in one well. Using one straight and one curved forceps, soak the gel-foam and tease the air bubbles out.
4. Place one filter on each gel-foam square.
5. Place the plate in a tupperware dish which has a dH20 soaked paper towl in the bottom of it. Pre-warm in the incubator.
Removing the lobes:
Have ready:
1. 2 petri dishes of cold PBS
2. 2 pairs of scissors, one fine
3. 3 pairs of forceps, one regular, one fine curved (Roboz # RS-5137), one ultra-fine (Roboz # RS-4905)
4. sterile 4x4 gauze
5. several 25g needles, both 1/2" and 1-1/2"
6. The FTOC culture dishes
Pregnant mothers are sacrificed by cervical dislocation or CO2. If using CO2, use the euthanization chamber on the 19th floor of Moos or use dry ice in a styrofoam box with tight fitting lid. Do not leave an open box of dry ice in a small or unventilated room.
The uterine sacs from each side, containing the embryos, are excised and placed in a petri dish with 10 ml of sterile cold PBS. From this point on everything should be performed in a horizontal laminar flow hood. (Never work with infected (viral or bacterial) tissues or cells in this hood.) Remove the individual embryos from the sac (using the fine curved forceps here helps). Next sever the placenta from each embryo and remove the sac tissue, place fetii in fresh cold PBS. Thymic lobes can be removed quite easily from a day 16 embryo. You need a dissecting scope and a good light source, preferably goose neck lamps. Place the fetus on top of a stack of gauze underneath the scope. Pin it thru the mouth and again at the base. Use another needle to tease the tissue above the thymus away. Pluck the lobes out using the ultra-fine forceps and place on filters. Be careful not to burst the thymic capsule at this point. Return to the tupperare dish and place in an incubator. Leave cover on tupperware box loosely. (It's my impression that humidity is pretty critical to how well the lobes develop.)
Feeding:
I replace the media in the cultures every day. To do this, carefully suction off the media with a sterile pastuer pipette. Add 1.2 ml fresh warm media.
7 days of culture is sufficient for good thymic development from a day 16 lobe.
Analysis
To disrupt the lobes, place on a small steel mesh , add media and crush the lobe against the mesh with the plunger of a 3 cc syringe. We can get 2-3 x10e5 cells per individually processed lobe this way. Yields go up to 5 x 10e5 cells per lobe if you pool them when harvesting.