Remove thymic lobes from the thoraxes of fifteen day old embryos (E14/E15) and trim exogenous connective tissue, using a dissecting microscope and fine watchmaker forceps. Place groups of 5-6 lobes on the surface of 0.45um pore size polysulfone filters resting on gelatin sponges, pre-immersed in RPMI-FCS-FTOC. Culture thymic lobes in 2ml of culture media supplemented with 1.35mM 2-deoxyguanosine for six days.
After culture, wash filters with thymic lobes, (X2) by immersion in 50ml of culture media for at least 2 hours at 37˚ C, 5% CO2-in-air. Remove lobes from filters and place in Terisaki plates, one lobe per well, containing 5 X 10^4 viable foetal liver cells and purified MTS mAb or rat IgG2a, at concentrations of 50-400 ug/ml, in 25ul of culture medium. Invert the Teresaki plates gently, to form a hanging drop, and incubate for 24 hours at 37˚ C, 5% CO2-in-air.
To prepare foetal liver cells, dissect livers from E14/E15 embryos and gently push through a 200 µm wire mesh sieve or between two frosted glass slides. Centrifuge the cell suspension (1500rpm, 7 min, 4˚ C) and resuspend the pellet in RPMI-1640 then gently layer over 5ml of ficoll-paque and centrifuge (1000rpm, 45 min, RT). Collect the pre-lymphocyte layer and wash by the addition of RPMI-1640 and centrifuge (1500rpm, 7min, 4˚ C). Adjust the cells to a final concentration of 4 X 10^6 viable cells/ml in culture medium.
After the 24 hour reconstitution incubation, wash thymic lobes in 5ml of culture media, to remove sticky cells, place on new polysulfone filters and gelatin sponges and culture for 18 days in culture media containing 50-400ug/ml purified MTS mAb or appropriate control eg rat IgG2a. Incubate cultures at 37oC in a humidified incubator containing 5% CO2-in-air for 18 days. Completely replace culture medium at Day 6 (D6) and Day 12 (D12) for 18 day cultures. NB The lobes can be re-wetted every 1-2 days with the petri dish media
At the termination of culture, carefully remove the thymus lobes from the filter discs using fine watchmaker forceps. Release cells were from the lobes using a small volume teflon-in-glass hand homogeniser. Place lobes in an homogeniser containing 500ul of cold PBS-FCS-Az and carefully disrupt to release the cells. Use an additional 500ul to rinse the homogeniser. The lobes may alternatively be crushed under a glass coverslip – scratch a cross in the bottom of a petri dish then put lobe(s) there, followed by 100 µL of media. Drop a coverslip on top of the lobe(s) and using a 2-10 syringe plunger press down on the lobe(s). Lift the coverslip and use 900 µL media to the wash it and the petri to recover as many cells as possible.
Cell viability and number can be assessed using ethidium bromide/acridine orange staining viewed with an Axioskop fluorescence microscope ( Zeiss, Germany) or a Coulter™ counter.
This additive for the FTOC media is for extra buffering and nutrients to last the culture period.
Use at 1/100 ie 5mL per 500mL bottle of media
Keep in foil (around the bottle) since folic acid is light sensitive, remember a famous man once said, ‘it is dark inside the body’
You need your antibody to be at 2mg/mL if possible, preferably dialysed into media (not essential)
in 35 mm petri dishes @ 3 mL ea
8-12 lobes per experimental group (4-6 lobes per disk)
6 embryos per group ~12 lobes (actually need 8 but best to overshoot ) = average per pregnant mouse
so 6 groups = 6 pregs I will order 10 and 10 more 6 days apart to arrive midweek
19 mL media @ each concentration = 6.65 mg total for 50,100 and 200 µg/mL - preferred. You can use half the amount of antibody if you have to by putting 2 disks in each petri dish.
For your final analysis you need to do a total 3 experiments.