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.
See also: FACs staining S.O.P. in this manual.
See also: PEC 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.
Prepare Antigen Presenting Cells:
Follow protocol for PEC preparation
1. Plate cells on sterile flat bottomed 96 well plates at 1x106/ml, 100ml per well in RP10 (1x105 cells/ml final).
2. Incubate at least 1 hour at 37oC to allow cells to adhere.
3. Wash the plate 4 times with RP10. (Add media, FLICK, add media, etc.)
Prepare Thymocytes:
1. Excise the thymus from a mouse euthanized by CO2 (cervical dislocation is not the preferred method here, since blood can accumulate near the thymus) taking care to get as little blood as possible.
2. Place the organ on a sterile steel screen with 5ml cold RP10 and press through with the rubber end of a 3cc syringe stopper to harvest the thymocytes. Collect cells and rinse once with another 5ml cold RP10. Wash 1x with cold RP10 and count. Resuspend cells at 1x107/ml in RP10. Keep cells on ice until ready to add to the plate. Add to plate at 50ml per well (5x105 cells/well final).
***NOTE: if either the PEC or the thymocytes are significantly contaminated with RBCs, you need to do RBC lysis prior to washing the cells and including them in the assay.
Prepare the Peptide and set up assay:
1. Peptide dilutions are in RP10 so should be done last, i.e. have everything else about the assay ready to go. Prepare the dilutions in separate tubes or plates, add the peptide to the wells with APC and thymocytes.
2. Incubate the assay plate at 37oC for 18-22 hours.
Stain the cells
1. Transfer the entire 200 ul to a round bottom 96 well assay plate, taking care to thoroughly resuspend the thymocytes.
2. Spin, flick, wash cells once in FACs buffer. Stain (follow FACs staining protocol) using bio 2.43 as the first step, CD4PE and SA-Fitc as the second steps.