Please find the following protocol:
Guidelines for the Culture of T Cell Lines and Clones
We work with two types of antigen specific T cell: either T cell hybrids, which are created by fusion of lymph node cells from antigen immunized mice to the thymoma BW5147, or T cell lines and clones which are established from the lymph node cells of antigen immunized mice. Whereas T cell hybrids proliferate continuously, and are thus available on a daily basis, T cell lines and clones are propagated by stimulation every 14 days with antigen, APC and IL-2 and are only available at the end of the 14-day cycle. Each has its advantages, but only T cell lines and clones can be used in adoptive transfer experiments.
The key to efficient cell culture is to plan ahead and prepare all of the reagents and components needed ahead of time. This can be done the afternoon before in the case of inert material such as 96 well plates that need to be labeled and formatted for proliferation assays and flasks that need to be labeled for restimulations. The labile components such as media and antigens should be prepared the morning the experiment is to be performed.
Minimize the length of time T cells are out of the incubator. It is best to avoid prolonged exposure to room temperature and levels of CO2 that are inadequate to maintain the pH of the DMEM. I suggest you follow the recommendations below and break the restimulation and testing into separate tasks. Do not take a significant break between the time you remove the old flasks from the incubator until you have placed the new flasks with the appropriate number of T cells (generally 5.0 X 105) for restimulation back into an isolator box and in the incubator and the cells for the proliferation assay on ice.
Prepare all of the BSS, DMEM/FBS/EL-4SN and other media you will need, such as non EL-4 supplemented DMEM prior to removing the T cells from the incubator. Prepare the
flasks needed for restimulation by writing the T cell designation, date, antigen and concentration on the new flasks. Tape these flasks together in the order that the old flasks have been set up. In addition, label a 15 ml centrifuge tube for each T cell line or clone for transfer of the cells required for a proliferation assay Another thing to consider is the stability of peptide antigens it is best to minimize their contact with warm temperatures.
Thaw peptides solutions in a beaker of warm water as soon as you take it out of the freezer. Prepare the antigens you need for the experiments prior to removing the cells from the incubator.
Once you have removed the T cells from the incubator work quickly, resuspend the T cells and take an aliquot for counting and count the samples immediately. After finishing the counts, determine the volume you will need for testing and for restimulation. Unless you require more than 2 ml of cell suspension to get enough T cells for restimulation, there is no need to pellet the cells. Therefore you can add the required volume of cell suspension to the pre-labeled flask for that T cell line or clone. Use the same pipette to transfer the number of cells you need for the proliferation assay to the pre-labeled 15 ml centrifuge tube.
When you have transferred the T cells to the new flasks, place both the new flasks and the old flasks back in the isolator box, gas, and place back into the incubator.
The tubes with cells to be used for the proliferation assay should be pelleted, the medium aspirated and placed on ice until you are ready for them. NOW YOU MAY TAKE A BREAK IF YOU WANT.
It is now time to prepare the spleen cells. I recommend that everyone wait until all T cell lines and clones to be restimulated are in the flasks. That way the spleen cells are added
when they are as fresh as possible. Estimate the number of spleen cells of each strain required that day and sacrifice the mice and take the spleens. I recommend that if more
than one strain of mouse is needed, each strain be done by a different worker. Take both strains at the same time and process the spleens and irradiate the cells promptly. As soon as they are back from the irradiator they should be added to the waiting flasks into which the T cells have been previously placed. The cells required for the proliferation assays should be placed on ice until use.