The overall goal of this experiment is to study the functional capacities of purified murine lymphocytes using various in vitro ad in-vivo functional assays. This method can help answer key questions in the fields of Immunology and Molecular Biology. Such as investigating function of a protein of interest in gene deficient lymphocytes.
The main advantage of this technique is the ability to quickly purify lymphocytes with minimal manipulation and environmental stress. To purify B cells re-suspend up to one times ten to the eighth red blood cell liced whole spleen cells in 300 microliters of balanced salt solution or BSS supplemented with FBS. Then add 50 microliters of anti-CD43 magnetic micro beads.
To remove the dead cells, add 30 microliters of Annexin five magnetic beads for 30 minutes at four degrees celsius. Flick the cell suspension at 15 minute intervals to ensure even labeling of cells. At the end of the incubation was the B labeled cells with 14 milliliters of BSS supplemented with FBS.
After centrifugation, remove the supernatant, flick the tube and re-suspend the pellet in one to three milliliters of room temperature BSS supplemented with FBS. During the centrifugation load a separation column onto a magnetic rack and attach a sterile 21 gauge needle to the tip of the column to reduce the flow rate. Equilibrate the column with two milliliters of room temperature BSS.
After re-suspending the pellet in one to three milliliters of room temperature BSS place the collection tube under the needle then load the B labeled cells onto the equilibrated column. Collect the flow through in a 15 milliliter conical tube. Then rinse the column with one milliliter of fresh BSS supplemented with FBS.
Pooling the wash with the collected target cells reload the column with the target cell containing aluet, collecting the flow through in the same 15 milliter tube. And wash the column three times with one milliliter of BSS supplemented with FBS, continuing to pool the washes with a target cell suspension. After the third wash, remove the column from the magnet and load it with five milliliters of BSS supplemented with FBS.
Using the column plunger to flush the magnetically labeled non target cells into a new 15 milliliter tube. Then, check the purity of the separated cells by flow cytometry. To re-use a separation column, after flushing the magnetic bead labeled cells wash the column three times from the top with five milliliters of PBS per wash and three times with five milliliters of distilled water per wash.
Next, wash the column with five milliliters of 70 percent ethanol in the same way and use an air tap to thoroughly dry the column before storage. To re-use the column in a new purification experiment re-wash the column from the bottom up with five milliliters of 70 percent ethanol followed by two five milliliter PBS washes. Then load the top of the column with five milliliters of PBS for one final wash.
And equilibrate the column with two milliliters of room temperature BSS. The column can then be loaded with the experimental cell population. To label the B purified cells with CFSE wash the cells two times with freshly prepared labeling solution in a 15 milliliter conical tube.
Re-suspending the second pellet at two times 10 to the seventh cells per milliliter concentration in fresh 37 degrees celsius labeling solution. Each time, centrifuge the samples remove the supernatant and flick the tubes. Then label the cells at a one part cell suspension to one part freshly prepared 10 micromolar CFSE solution ratio in the dark for 10 minutes at 37 degrees celsius.
Inverting the tube every two minutes to ensure an even distribution of the dye. Caution should be taken to ensure that the cell pellet is thoroughly suspended in a labeling solution. Cell clumps can affect the homogeneity of the CFSE labeling.
At the end of the incubation add RPMI medium to quench CFSE and wash the cells two times in several volumes of ice cold complete RPMI medium. If the cells have been successfully labeled the pellet will be yellow in color. Next, wash a 96 well flat bottom plate coated with the appropriate stimulus two times in PBS.
For CFSE labeled B cells, re-suspend to three times 10 the sixth B cells per milliliter and complete RPMI medium and seed 100 microliters of B cells per well onto the coated plate in triplicate for 72 hours. By this depletion method the purity of the bead isolated OT1-CD8 T cells can be increased from 72.8 percent to 94.2 percent. The cells can then be labeled with CFSE as just demonstrated and adoptively transferred into recipient animals for analysis of their in-vivo proliferation.
In addition, anti-CD45.1 antibody labeling can be used to further confirm the identity of the CFSE expressing cells as the adoptively transferred donor CD-45.1 positive OT1-CD8 T cells in the lymphoid organs of recipient mice. Alternatively, the purified T cells can be stimulated in vitro and their proliferation can be assessed by tritiated Thymidine incorporation. A significant increase in splenic B cell purity is also achieved by this method.
Facilitating similar opportunities for downstream functional analysis of the purified B cells. Once mastered, this technique can be completed in two hours. By performing this procedure, it is important to keep your CI suspension on ice at all times except during purification and CFSE labeling.
Purified lymphocytes can also be used in other functional assays such as immunoblotting to the terminus signaling capacity of genetically modified lymphocytes. After watching this video you should have a better idea of how to purify B and T cells and how to use the cells for various in vitro and in vivo functional assays.
