The overall goal of this procedure is to use an in vitro human suppression assay to measure treg function and to potentially anticipate future loss in their function. This is accomplished by first preparing and testing coded beads, which will be used as cell stimulators. The second step is to isolate pure naive CD four positive CD 25 negative T cells in vivo activated CD four positive, CD 25 low T cells, and regulatory CD four positive CD 25 high T cells N to a radiate PP BMCs for use as antigen presenting cells or APCs.
Next single cultures are set up of naive or activated T-cells as responders with P BMCs as APCs or co cultures under the same conditions, but with tregs added at a one to 10 ratio. The final step is to harvest the cells and measure their proliferation by tritiated. Thymidine incorporation ultimately obtained results can demonstrate differences in the suppression of naive versus in vivo activated T-cell responses.
My name is Jeff Woodcliff. Regulatory T cells do not express unique cell surface markers. Therefore, in order to reliably measure their function, they must be isolated using techniques, giving the best separation.
The main advantage to using fluorescence activated cell sorting over other methods like max isolation, is that fax offers the pure cell populations that can be achieved. Cell function can then be reliably tested in in vitro suppression assays as demonstrated here Though, this method can provide insight into the treg function in healthy and subjects related to type one diabetes. It can also be applied to other autoimmune diseases such as human rheumatoid arthritis, systemic lupus, ery mitosis, multiple sclerosis, and others To coat taal activated beads with anti-human CD three.
Add one milliliter of the M four 50 toil activated beads from the original vial into a 1.5 milliliter tube, and place the tube in a magnetic stand until all the beads have adhered to the side of the tube. Then remove the bead buffer by pipetting it out while the tube is still in the magnetic stand. Then take the tube out of the magnetic stand and add one milliliter of buffer one.
Place the tube in the magnetic stand again and again. Remove the buffer while the tube is in the magnetic stand. Resus, suspend the beads in one milliliter of buffer one again.
This time adding 40 microliters of anti-human CD three as well. Agitate the beads and antibody at 37 degrees Celsius for 15 minutes. Then add 0.1%weight per volume BS, A, and continue agitating for the next 16 hours.
Next, wash the beads twice with buffer two by incubating the beads in one milliliter of buffer. Two for five minutes at two to eight degrees Celsius, and then removing the buffer while the tube is in the magnetic stand. Then wash the beads one more time under the same sterile conditions.
This time using one milliliter of buffer three, test the efficiency of the antibody coating by adding a variable number of CD three coated beads per cell in order to determine the optimal ratio and ALI quoting 50, 020 5, 000 P BMCs per well in triplicate in a 96 ball plate. After 72 hours in culture at 37 degrees Celsius, add one micro curry of tritiated thymidine and continue incubating for the next 16 hours. Harvest the cells in a multi-screen harvest plate or similar.
Add scintillation liquid and read the counts per minute or CPM per well. Using a top count N XT scintillation counter, or similar, determine the bead to cell ratio using the one giving A CPM consistently above 5, 000, but less than 15, 000 in our experience. A ratio of three beads per cell usually gives the optimal stimulation for both responder and treg cells in all human subjects tested.
Fill a 50 milliliter conical tube with 15 milliliters of fal plus. Then perform a one to six dilution of 50 milliliters of buffy coat by adding 250 milliliters of PBS to the tube of blood. Slowly layer 25 milliliters of the diluted buffy coat on top of the fial pack without disturbing the layers.
Centrifuge at 800 times G for 30 minutes at four degrees Celsius. With the break turned off carefully collect the PBMC layer, which is the white intermediate phase between fial and the buffer. If needed, scrape cells from the wall of the tube.
Wait a few minutes and collect them from the intermediate phase Again. Transfer the cells to fresh 50 milliliter tubes and wash them by filling the tubes with PBS pellet. The PBM cs.
Repeat the washing step twice each time. Combining two cell pellets into a single tube, ultimately combine all the cell pellets into a single 50 milliliter tube. Then use 20 microliters of the suspended cells to determine the cell viability by trian blue exclusion.
Test generate APCs by transferring one milliliter of the viable pbmc into a new tube. Then add four milliliters of media and irradiate the cells with 5, 000 rads top off the tube containing the rest of the P BMCs with PBS buffer. Then after spinning the cells at 250 times G for 10 minutes at four degrees Celsius, pour off the supernatant and resuspend the cells in four milliliters of the same admixture.
Then add 200 microliters of max anti CD four microbeads to the pbmc and incubate the cell and bead mixture at four degrees Celsius for 20 minutes. After the incubation, wash the cells in 40 milliliters of PBS buffer, then pellet the cells. Then pour off the supernatant and resuspend the cells in eight milliliters of Degas PBS buffer at room temperature.
Next, use pres separation filters to filter separate the cell suspension. Then divide the PBM CS into two 15 milliliter tubes. Adding four milliliters of the P BMCs to each tube.
A thick affix two s columns to a minimax separator and place collection tubes below each column. Then add three milliliters of DGAs PBS buffer to each column when no buffer is left layer the entire four milliliter cell solution from each tube over the columns before the cell suspension completely runs out. Add three milliliters of DGAs room temperature PBS buffer to each column and let the buffer run through into the collection tubes.
Add more buffer until it comes out clear. Pipette five more milliliters of the DGAs buffer onto the LS columns, and then remove the LS columns from the minimax separator. Place the columns into new sterile 50 milliliter collection tubes.
Let a half milliliter run out and then slowly plunge the rest of the volume out, which is about four milliliters. Now combine the two CD four positive fractions. Then add up to 50 milliliters of PBS to the tube and count the cells after spinning the cells at 400 times G for 10 minutes at four degrees Celsius.
Resuspend the cells in two milliliters of PBS buffer. Combine the antibodies to be used for fact staining into one tube anti CD 14, CD 32, CD one 16 and optionally anti CD four. Take an aliquot of mixture for staining the FLUOROCHROME minus one tube.
Prepare the FMO tube by transferring five microliters of the tube milliliter cell suspension to a fax tube and stain the cells with two microliters of the previously prepared antibody cocktail. Next, add unstained cells and beads coated with mouse IgG to be stained with a single fluorochrome to new individual fax tubes for use as compensation controls for cell sorting. Then add 50 microliters of anti-human CD 25 PE to the cocktail and add the cocktail to the rest of the cell suspension.
Incubate the tubes at four degrees Celsius for 30 minutes. After incubating the cells with the antibody cocktail, add PBS buffer to the cells and then pellet them Resus. Suspend the pellet in PBS buffer at a concentration of 10 to the seven cells per milliliter leader.
Use the FMO tube to set the threshold for sorting of CD 25 positive T cells. Using this gating strategy, first, set a gait around the fite negative cells in order to exclude the fitz positive cells, which comprise monocytes, macrophages, and all other CD four positive non T cells as demonstrated in this figure. In a separate plot, draw gaits for CD 25 negative and CD 25 low T cells and CD 25 high treg cells as in this figure.
After flow, sorting the cell subsets, pellet the collection tubes and then keep the T-cell on ice until plating. Label a U bottom 96 well plate as shown in the table. Suspend a 50 microliter aliquot of CD three coated beads calculated to a three bead per responder cell per well concentration in complete media with 10%pooled human AB serum in the 96 well plate then dilute the previously irradiated APCs in media to a concentration of five times 10 to the fifth cells per milliliter.
Add 50 microliters of the cells to the 96 well plate, and then add 2.5 times 10 to the four CD four positive CD 25 negative or CD four positive CD 25 low T-cell per well in triplicate add tregs to row B and to the wells labeled as tregs only of the co-culture in a one to 10 ratio as shown here. Then incubate the plate at 37 degrees Celsius in an incubator with 5%CO2 in saturated humidity for 72 hours. Next, pulse the cells with one micro curie of tritiated thymidine and then continue the incubation at 37 degrees Celsius for the next 16 hours after the thymidine has been incorporated in the last few hours of the co-culture.
Use a filter mate harvester to harvest the cells on a multi-screen harvest plate. Then cover the harvesting plate with a transparent plastic cover previously filled with a scintillation fluid. Finally read the CPM per well.
Using a scintillation counter T-cell subsets can be isolated to a high purity by this system as seen in the following series of dot plots CD four positive CD 25 negative T cells are shown here in the bottom gate. In this next figure, highly purified CD four positive CD 25 low T cells can be seen here. A pure population of flow sorted CD four positive.
CD 25 high treg cells is shown in the top gate control. Single cell type cultures of APCs and tregs from healthy subjects exhibited little to no proliferation over background as measured by thymidine incorporation, whereas naive CD 25 negative and in vivo activated CD 25 low T cells exhibited marked proliferation that is suppressed when these responder cell types are co cultured with treg cells. Although slight differences in the capacity of tregs to suppress responder naive CD 25 negative and in vivo activated CD 25 low T-cells was observed.
It was not significant control. Single cell type cultures of APCs and Tregs from at risk subjects exhibited little to no proliferation over background as measured by thymidine incorporation. Whereas naive CD 25 negative and InVivo activated CD 25 low T cells exhibited lower levels of proliferation compared to healthy controls.
The responder T cells were further suppressed by tregs and co cultures since the assay generates reliable results. We can additionally conclude that low CPM values suggest potential defects in signal transduction, which should be further investigated in a different type of study. The difference in capacity of tregs to suppress CD 25 negative versus CD 25 low responder T cells in at risk subjects however was significant.
After watching this video, you should have a good understanding of how to smoothly connect all the necessary steps for a successful and reliable functional test for the presence of Tregs in healthy subjects and subjects with compromised immune systems. For example, in subjects with type one diabetes.
