Type one diabetes is an autoimmune disease in which insulin secreting pancreatic beta cells are destroyed by autoreactive T cells. Understanding the mechanisms involved will provide insights to generate an effective cure for this disease. Here two methods to observe specific cd eight positive T-cell mediated destruction of pancreatic beta cells in vitro are demonstrated a chromium 51 based cell mediated lympho cytotoxicity or CML assay and a visual CML assay for both methods.
CD eight positive T effector cells are first prepared for mouse spleens to perform A CML assay knit one or knit four target beta cells are labeled with chromium 51 and co cultured with a effector cells. When target beta cells are killed, the chromium 51 is released. The supernatants and cell lysates are then collected and radioactivity levels which indicate the extent of cell death are measured using a gamma counter to perform a visual CML assay Knit one and knit four Target beta cells are labeled with mitochondrial potential dye TM RM effector cells are labeled with pico green.
The cells are combined and live cell imaging is performed to examine the rate of cell death indicated by dissipation of mitochondrial membrane potential. The main advantage of this assay over traditional assays such as the chromium release assay, is that we can observe beta cell death in real time. Additional advantages include the ability to determine whether CD eight T cell mediated death of beta cells is contact dependent and also this assay can be extended using fluorescent indicators to map out the pathways of beta cell death.
This method can answer key questions in the pathogenesis of any disease where cytotoxic T cells play a role. The implications of this technique extend towards therapy and possibly diagnosis because mitochondrial function is essential in both beta cell survival and physiology. Jing Chen and I will demonstrate this procedure.
The target cells used in this demonstration are the mouse beta cell lines, knit one and knit four, the jerk at CD three plus human lymphocyte cell line, which does not recognize and kill mouse beta cells is used as a negative control the effective cells used. Here are Autoreactive CD eight positive T cells obtained from the spleens of three to four week old nod AI four alpha beta F1 mice to obtain the cells begin by placing the spleens in a seven milliliter glass down homogenizer containing five milliliters of ice cold HBSS homogenize the spleens using a downs homogenizer then transfer the cell suspension to a conical tube and centrifuge at 250 Gs for five minutes following the spin, discard the snat then to remove the red blood cells, resuspend the pellet in five milliliters of hypotonic solution per spleen. Incubate for five minutes on ice.
After the incubation wash the cell pellet twice with 10 milliliters of ice cold HBSS centrifuge. The cell suspension between each wash resuspend the cells in 10 milliliters of ice cold HBSS. Then using a hemo cytometer count the cells after the cell counts have been determined.
Centrifuge again then resuspend the cells and supplemented RPMI 1640 at a density of five times 10 to the six cells per milliliter seed cells at a density of five times 10 to the six per milliliter in a T 75 flask. Finally, to activate the cells add AI four myope and IL two to the flask at final concentrations of 0.1 micromolar and 25 units per milliliter respectively incubate the flask in a 5%CO2 incubator at 37 degrees Celsius for three days. The activated cells can now be used for either ACROMIUM 51 based CML assay or a visual CML assay.
The cell mediated lympho cytotoxicity or CML assay here uses the release of chromium 51 from target cells as an indicator of CD eight mediated beta cell death. To label knit one or knit four target beta cells with chromium 51 begin by seeding them at a density of five times 10 to the fourth cells per well of a 96 well plate in 200 microliters of supplemented DM E EMM incubate at 37 degrees Celsius 5%CO2 for 24 hours. The next day dilute chromium 51 and supplemented DMEM to a concentration of one micro curie per 20 microliters.
Next, add 20 microliters of the diluted chromium 51 to each well containing the target beta cells to achieve one micro curie per well. Place the plate in a humidified 37 degree Celsius incubator with 5%CO2 and incubate for three hours following the incubation. Aspirate the medium and wash the cells three times with fresh culture medium.
Next, add medium to the activated CD eight positive effector cells such that the final volume added to each well will be 200 microliters. Combine the effector in target beta cell cultures at desired E to T ratios here, ratios of 20 to one, 10 to one, five to one, two to one, one to one and 0.25 to one are used to prepare spontaneous lysis controls. Add fresh medium to six wells of the target beta cells.
Then place the plate in the incubator and co-culture effector and target cells for 16 hours. The next day use a multi-channel pipette to transfer the supernatant to six times 50 millimeter lime glass tubes. Then to lyce the cells at a hundred microliters of 2%SDS to the plate and gently pipette up and down several times.
Transfer the cell lysate to a new set of six times 50 millimeter lime glass tubes. Then to ensure that all the cell lysate is collected at a hundred microliters of sterile water to each well pipette up and down and add it to the cell lysate tubes to determine the degree of beta cell destruction. Measure the radioactivity in the supernatant and lysate using a gamma counter once the counts have been performed.
Determine the specific lysis by subtracting the percentage of counts in spontaneous lysis Control supernatants from the percentage of counts in the experimental supernatants as shown in this equation. To examine T-cell mediated beta cell death in real time. Mitochondrial membrane potential is monitored by live cell imaging of target and effector co cultures.
To prepare for microscopy, see target beta cells on modified and sterilized eight well chambered cover glass at five times 10 of the fourth cells were. Well place the slides in an incubator at 37 degrees Celsius and 5%CO2 and allow the cells to grow for 48 hours. On the day of the experiment, add the mitochondrial membrane potential dye tetraethyl rumine methyl ester TMRM at 25 nanomolar and phenol red free supplemented DM EM incubate for 30 minutes.
Once the cells have been stained with TMRM, replace the medium with fresh medium containing five nanomolar TM RM to maintain the equilibrium distribution of the dye. Next count the activated effector AI four T cells or control jerk it cells using a hemo cytometer resuspend the cells in phenol red free supplemented DMEM in 15 milliliter conical tubes at the appropriate concentration. Next, add pico green to the activated effector cells or jerk it control cells to a final concentration of one microliter per milliliter.
Wrap the tube in aluminum foil to protect it from light, then incubate for five minutes at room temperature following the incubation. Wash the cells three times by adding phenol red free DMEM mixing briefly and centrifuging at 250 GS for five minutes. To collect the cells for imaging, place the chamber slide on the stage of a confocal microscope equipped with a motorized stage and a humidified live cell imaging chamber at 37 degrees Celsius and 5%CO2.
Add the activated pico green stained effector cells to the chambers at the effector to target ratio to be assessed here. The ET ratio imaged will be 50 to one set up the imaging software to collect images in four wells with three locations each every three minutes with 300 frames per location and ex citation using the appropriate filter sets for TMRM and Pico Green. Once all of the images have been collected, they must be converted for publication here.
Fiji and Image J are used with a bio formats plugin from loci. More details regarding video conversion can be found in the accompanying written text. A cell mediated lympho cytotoxicity or CML assay was performed using chromium 51 as described in this video.
As shown here, the percent specific lysis shown in the Y axis increases as the effector to target ratio shown in the x axis increases to assess cell death by live cell imaging, the mouse beta cell line knit one was stained with a mitochondrial membrane potential dye TMRM shown in red activated autoreactive I four CD eight positive T cells were staying green with pico green and the two cell types were co cultured at an E to T ratio of 50 to one as can be seen here. Knit one mitochondrial membrane potential dissipated gradually during a 400 minute duration, but only in the clusters that we're interacting with green AI four T cells as a control experiment. Stained knit one cells were instead cultured with pico greens stained MHC mismatched human jerk at T cells as can be seen here.
Knit one cells were able to maintain mitochondrial membrane potential throughout the duration of 400 minutes. Circuit cells did not interact with knit one clusters and therefore did not induce killing. Once mastered.
This technique takes approximately five days including cell preparation. Once observing this video, you should have a good understanding of how beta cell membrane potential changes when under attack by Autoreactive T-cell. Don't forget that working with radioisotopes can be hazardous and the proper training should be obtained and precautions taken while performing this procedure.
