Hi, I'm Andrea White and I'm from Graham Anderson and Eric Jenkinson's lab at the MRC Center for Immune Regulation at the University of Birmingham. Today I present a protocol for deriving re-aggregate thymic organ cultures, which can be used to study complex cellular interactions which occur during T-cell differentiation. Prior to the start of this procedure, embryonic thymic lobes were isolated and treated to remove all lymphocytes.
This can be seen on a separate video. The alienoid lobes are then disaggregated to produce a stromal cell preparation. And finally, the cells are aggregated to form a re-aggregate fmic organ culture.
Let's begin. After five to seven days in culture, the LO should be ready to disaggregate for shoal cell preparations submerge the 0.8 micrometer filters in a Petri dish containing RPMI 1640 plus 10%FCS Using forceps gently push off any fiber slopes that do not float off. Use a one mil per pet to transfer the lobes to a 1.5 mil eend orph tube.
We recommend no more than 50 lobes per eend orph tube. Allow the lobes to settle to the bottom of the tube under unit gravity and remove the medium. Wash the lobes with one mil PBS and then let the lobes sink to the bottom again.
Repeat this for a total of three PBS washes, remove the last PBS wash and add 600 microliters of a 0.25%trips in solution. Place the einor tube in a 37 degree incubator for 25 minutes. Gently tritrate the lobes using a one mil blue tip on a perpet for 10 seconds, and then place back in the incubator after 20 minutes, pipet the lobes up and down to completely disaggregate them.
It should take about 30 seconds To achieve a single cell suspension, add 400 microliters ice cold, RPMI 1640 plus 10%FCS to neutralize the trypsin imp pellet the cells by centrifugation at a thousand RPM, 10 minutes at four degrees C, remove the sup natant and resuspend the pellet in one mil. RPMI 1640 plus 10%FCS if desired. Count cells.
Typically we would expect around 50, 000 cells per dioxazine lobe, starting from an E 15 fibrous. Now we are ready to form the aggregate fmic Corgan cultures in a one and a half mil eend orph tube. Mixed, freshly prepared fine stromal cells.
Together with pre-selected CD four positive CD eight positive fibrocytes. We recommend about a one-to-one ratio of stromal cells to fibrocytes. The number of cells you will use will depend on the number of cells available and the experiments to be performed.
We recommend at least a hundred thousand cells in total and no more than a 1.5 million cells. You want the V aggregates to be large enough to be reproducible, but not so large that the center will die pellet cells by centrifugation at a thousand RPM for 10 minutes. At four degrees C, assemble the organ culture filter carefully.
Remove all a SUP natin in stage with a P 200 per pet. The vortex cell pellet for 10 seconds to form a slurry. The volume should be around one to two microliters mouth.
Pet the slurry into a finely drawn pet, keeping the suspension of cells at the tip of the pet. Under our dissecting microscope, carefully mouth PET the cell slurry onto the center of a 0.8 micrometer filter. Ideally, cells should be placed as a standing drop in a circle of one to two millimeters in diameter.
If the slurry begins to spread out, allow the fluid in the slurry to drain through the pos before depositing the remaining cells. Place dish in a plastic sandwich box and seal. As for thymus organ cultures, and place a box in an incubator, we aggregate cultures form into intact structures within 18 hours of culture.
After four to five days of culture, they will generate both CD four positive eight negative and CD four negative eight positive mature cyte subsets. Starting with 50 dioxazine treated lobes should give enough stromal cells to prepare three to five reaggregate cultures. These cultures typically appear as coherent mounds of cells that can be pushed off the filters using forceps without disrupting them.
We have just described the method in which to study positive selection by F epithelial cells. This method can also be used to study dendritic cell mediated negative selection. Finally, by using TCR transgenic FIBROCYTES or MHC Class one deficient or MHC Class two deficient stromal cells.
This system allows separate investigations into CD four positive or CD eight positive T-cell selection. Thanks for tuning in.
