This protocol allows us the ability to isolate lymphocytes T-cells from patients with the disease for studies, the molecular defect and immunologic defect. This method allows us to isolate viable cells, cells with the disease abnormalities for purification of RNA, DNA, and protein from these patients and that allows us to characterize molecular defects to understand pathways abnormalities that are important in pathogenesis. This method is valuable for isolation of peripheral blood that can be further fractionated to study blood cells in normal states, as well as blood cells from diseases with immune abnormalities.
Demonstrating this procedure will be Alanna, who is a medical student research fellow from my laboratory. After obtaining a peripheral blood sample in five 10 milliliter tubes containing anticoagulant, transfer 10 to 15 milliliters of blood into 50 milliliter separation tubes labeled with the subject number and dilute the samples at least twofold, but to no more than 35 milliliters per tube with HBSS. Carefully and slowly underlay the blood with approximately 13 milliliters of density medium per tube, stopping the pipetting when the pipette is almost empty to prevent bubbles.
Remove the pipette slowly to avoid mixing the blood and density medium layers and carefully transfer the filled separation tubes to the centrifuge without disturbing the layers. After separating the cells by centrifugation, remove all but the last 10 milliliters of the plasma containing upper layer, before carefully and slowly collecting the buffy coat. Pool the buffy coat from between two separation tubes into a single labeled sterile 50 milliliter tube and dilute the PBMC at least twofold with fresh HBSS to a total of 50 milliliters per tube.
When all of the buffy coats have been collected, pellet the cells by centrifugation and remove as much of the supernatant from each tube as possible. Tap the tube bottom to loosen the pellet and resuspend the pellets in one to two milliliters of ACK lysis buffer per original 10 milliliter blood sample volume per tube. After exactly five minutes, stop the lysis in each tube with an equal or greater volume of HBSS and adjust each volume to 50 milliliters.
After centrifugation, discard the supernatant, pool the cells from the same donor and bring volume up to 50 milliliters with fresh HBSS for another centrifugation. Then resuspend the cells in 10 milliliters of 37 degree Celsius RP 10F medium for counting. For CD4+CD45RO+T-cell purification, spin down the cells and dilute the PBMC to a five times 10 to the seventh cells per milliliter of selection buffer concentration.
Transfer the cells to a five milliliter round bottom polystyrene tube and add 50 microliters of antibody cocktail per one milliliter of sample with gentle mixing. After five minutes at room temperature, vortex magnetic particles at high speed for 30 seconds before adding 50 microliters of magnetic particles per one milliliter of sample to the tube with gentle mixing. Bring the volume to 2.5 milliliters with fresh selection buffer and gentle mixing and place the tube onto a magnet for 2.5 minutes.
At the end of the incubation, pick up the magnet and invert the tube in one continuous motion to decant the enriched cell suspension into a new sterile tube. To increase the cell recovery, add 2.5 milliliters of selection buffer to the tube without disturbing the immobilized beads for another 2.5-minute incubation on the magnet and add the wash to the collection tube, then count the number of viable cells using a hemocytometer and confirm the cell purity by flow cytometry. To activate the cells, adjust CD4+CD45RO+T-cells to a five times 10 to the sixth cells per milliliter of 37 degree Celsius RP 10F medium concentration and seed the cells onto suitably sized culture dishes.
Rest the cells in a humidified 37 degree Celsius, 5%carbon dioxide incubator overnight. The next morning, collect the rested cells by centrifugation and resuspend the pellet at a five times 10 to the sixth cells per milliliter of 37 degree Celsius RP 10F medium concentration. Split the suspension into five to 10 times 10 to the sixth cell aliquots in each of three sterile screw cap tubes and stimulate the cells in tube two with PMA and tube three with A23187 and gentle mixing.
Add an equal volume of dimethyl sulfoxide to the first tube to serve as the vehicle control and place the tubes with the caps loosened into the incubator for the appropriate activation period. At the end of the incubation, collect the cells by centrifugation and discard as much of the supernatant as possible without disturbing the cell pellet. Then lyse the cells as directed by a commercially available RNA isolation kit and isolate the RNA according to the manufacturer's instruction.
The viability and purity of CD4+CD45RO+T-cells obtained by this negative selection protocol is consistently high. The yield of CD4+CD45RO+T-cells obtained from leukoreduction system chambers from SS PBMCs is typically about 75%compared to about 16%from normal donor PBMCs. SS memory T-cells and SS PBMCs poorly expressed cytokine and other immune response genes compared to cells from normal donor T-cells and PBMCs.
In addition, many genes not normally expressed in normal donor T-cells are highly expressed in SS T-cells both at rest and following stimulation. Good sterile technique and a consistent routine are really important for the success of this technique. It's also really important while you're layering the Ficoll that you use an automatic pipette with fine control and you really pay attention to the speed that you're laying down the Ficoll.
Cells isolated using this method will be viable and can be used for toxicity studies, molecular biochemical studies for characterization of signal transduction pathways, flow cytometry and gene expression studies. The method can be used for isolating cells from patients with disease, for inflammatory diseases, and also other diseases such as cancers for direct studies. Examples of these diseases include psoriasis and atopic dermatitis.
