Fetal Liver Reconstituted FTOC
50mL FCS (10%v/v)
* Make up 2000X stock of mercaptoethanol by taking 35uL of mercaptoethanol from bottle (which is at 14.3M) into 5mLs of RPMI (this is a 1/143 dilution to give a 0.1M solution, or 2000 X stock). 250uL of this into 500mL FTOC media gives working concentration of 50uM (5x10^-5M).
** Weigh 0.018g of deoxyguanosine and transfer into 5mLs of FTOC medium (this gives a 10X stock; 13.5mM). This will initially look very cloudy, so wait until it dissolves fully before filtering. Sterile filter this 10 X stock into 45mLs of FTOC medium to give working concentration of 1.35mM.
Best to setup the FTOC plates the day before the embryos are ready to ensure sterility. Into 6 well plates add 2-3mLs of medium/well. Cut squares of gelfoam into petri dishes full of FTOC medium and mash sponges to wet completely. Place one square/well, then top with sterile polysulfonate filters. Wet surfaces of filters just prior to putting on lobes.
Remove thymic lobes from E14/15 embryos under dissecting microscope in laminar flow hood. Remove exogenous tissue using fine watchmaker forceps. Place groups of 6-8 lobes on the surface of wetted polysulfonate filters resting on gelfoam sponges. Culture in 5% CO 2-in-air incubator for 5-7days. Lobes will not look a great deal different as the FTOC proceeds, but should be noticeably flattened once harvested.
To harvest dGuo treated lobes, gently wet the filter and place near/in media (without gGuo) in a petri dish. Then with fine forceps, carefully roll the lobes off into the media, without grasping the lobes at any stage. Use one forcep tine to gently work the lobes off the filter one by one. Harvest all lobes from petri dish using glass pipette (careful not to let lobes go beyond widened neck of pasteur pipette, otherwise they may stick inside pipette) and transfer into 50mLs of media in tube and put back into incubator. Allow to wash for 1 hour, then change media (leave lobes settled on bottom) and wash for another hour. This is to ensure that no dGuo is left on the lobes.
Meantime, prepare fetal liver cells by dissecting from E14/15 embryos. Gently push through sieve, or between sterile frosted glass slides into medium. Centrifuge cells (1500rpm, 7min, 4 oC) and resuspend in 3mLs of RPMI and gently layer over 5mLs of Ficoll-Paque. Centrifuge at 1000rpm for 45mins at room temperature without brake. Collect the buffy layer, DO NOT take any of the clear bottom layer (Ficoll-Paque). Count cells and adjust to 2 x 10^6 viable cells/mL of normal FTOC medium (no dGuo). Plate out 25uL/well of a Terisaki plate (5 x 10^4 fetal liver cells/well). Place one lobe/well then invert plate for culture.
Reconstitute for 24 hours in 5% CO 2-in-air incubator then wash lobes in 5mLs of culture media to remove sticky cells, then place on new filters. Culture for another 18 days, changing media every 6 days. Wet the lobes with media from wells every second day.
Carefully roll the lobes off the filters using watchmaker forceps into small petri dish with 200uL media. Gently crush lobes under a glass coverslip using a small syringe plunger to push down. Lift the coverslip and use 800uL of media to wash it and the petri dish to recover cells. Count cells under haemocytometer with acridine orange/ethidium bromide to distinguish viable and dead cells.