The overall goal of this procedure is to isolate and infect primary as in our cells for the purpose of measuring cytosolic calcium levels and cell injury. This is accomplished by first isolating home mouse, pancreas tissue, and mincing the tissue in collagenase buffer. In the second step, the mince tissue is briefly incubated in collagenase buffer at 37 degrees Celsius.
Next, the cells are filtered and then resuspended. In culture media. In the final step, the cells are plated for cell injury, calcium measurements, or adenoviral infection.
Ultimately, LDH leakage, subcellular, calcium fluorescence, and adenoviral. Fluorescence can be measured to determine cell toxicity changes in calcium levels and adenoviral infection efficiency respectively. The main advantage of this technique is that it provides a sensitive method by which to measure ASIN or cell cytotoxicity and calcium signals.
In addition, it allows for reliable infection of pancreatic our cells with adenovirus. The implications of this technique extend toward the therapy of acute pancreatitis because it allows for rapid and thorough screening of potential biochemical pathways that may play critical roles in the pathogenesis of this disease. Visual demonstration of this method is critical since the cell preparation and calcium measurement procedures require a delicate and precise technique as well as some experience with confocal microscopy Before starting the experiment.
First, immerse 22 by 22 millimeter cover slips in two parts nitric acid and one part hydrochloric acid. After two hours, decant the slides with the ionized water. After euthanization, orient the animal in the subline position and prepare the abdominal surface by cleaning with 70%ethanol.
Perform a laparotomy to expose the abdominal cavity, dissect out the pancreas, and immediately rinse in a small whey boat containing six milliliters of collagenase buffer and dissect away any large pieces of fat or visible blood vessels. Then remove five milliliters of collagenase digestion buffer and add it to the 15 milliliter conical tube. After removing all but one milliliter of collagenase use fine dissection scissors to mince the pancreas until the resulting solution appears evenly dispersed.
Then transfer the mist product to a 125 milliliter erlenmeyer plastic flask. Shake the flask in a 37 degree Celsius water bath at 90 RRP M for 30 minutes. During this time, rinse the 22 by 22 millimeter acid washed cover slips with the ionized water.
Then dry the cover slips and place them on top of a flat surface lined by laboratory film. When the 30 minute digest is complete, transfer the suspension back to the 15 milliliter conical tube. After allowing the cells to settle carefully, pipe it out the collagenase digestion buffer and replace it with six milliliters of freshly prepared bovine serum albumin incubation buffer.
Then vigorously shake the tube by hand for 10 seconds in order to disperse the cells into smaller clusters. Immediately after shaking, remove any large floating debris from the unsettled media. After allowing the remaining cells to settle, exchange the media with fresh BSA incubation buffer.
Then remove the BSA incubation buffer and replace it with freshly prepared heaps incubation buffer. Now dilute the calcium dye to a final concentration of 7.5 micromolar in freshly prepared BSA free heaps incubation buffer, and then plate 500 microliters of this suspension onto each of the 22 by 22 millimeter cover slips. To measure the calcium concentration in the pancreatic ASIN R cells begin by using fine tweezers.
To carefully grasp one cover slip containing cell suspension at its edge, tilt the cover slip at a 45 degree angle, allowing the excess buffer to run off. Then place the cover slip on top of the rubber gasket with the cells on top of the cover slip and assemble the chamber. Next, secure the perfusion chamber to the stage and insert the tubing containing the buffer into the first inlet of the chamber.
Turn on the syringe containing the buffer and allow the buffer to perfuse over the entire surface of the cover slip. Once the buffer has reached the opposite end of the chamber, insert a vacuum line into the vacuum inlet. Then to visualize the cells using either a 200 x or a 400 x 1.4 numerical aperture objective and an argonne laser excite the flu oh 4:00 AM die at a wavelength of 488 nanometers.
To prepare the pancreatic asar cells for the cell injury assays begin by transferring the pancreas to a clean way dish. After mincing and digesting the tissue as just demonstrated, remove the flask and allow the cells to briefly settle. Then replace the snat with five milliliters of fresh collagenase buffer.
After shaking the tissue for an additional 35 minutes, use a transfer pipette to vigorously resuspend the digest until the suspension appears homogeneous with no obvious cell clumps. Next, pipette the cell suspension through a pre-wet nylon mesh into a conical flask. Then use an additional three milliliters of fresh D-M-E-M-F 12 media with collagenase to vigorously pipette the suspension through the mesh to force through any remaining cells.
Now add another six to 10 milliliters of D-M-E-M-F 12 media to the flask. After Resus suspending the cells in fresh D-M-E-M-F 12 media plate 500 microliters of the cell suspension into each well of a 48 well tissue culture plate. The cells should resemble the cells in these representative images.
After allowing the plate to shake in the water bath for another five minutes, stimulate the cells with varying agonists. After two to four hours, carefully remove and flash freeze a 100 microliter eloqua of the cells in liquid nitrogen. To measure the lactate dehydrogenase leakage or LDH first, we constitute the LDH substrate that is provided in the kit with 12 milliliters of the provided assay buffer.
Then warm the frozen cell suspension and media samples in a water bath at room temperature. When the samples are thawed plate 50 microliters of each media sample into a 96 well plate now dilute the cell suspension from 10 x lysis buffer to a final concentration of one x. Then vortex the cell suspension briefly and incubate the cells at 37 degrees Celsius for 60 minutes.
Next plate, 50 microliters of the one to 10 diluted lysed cells into each well of a 96. Well plate add 50 microliters of reconstituted substrate to each well and incubate the plate in the dark at room temperature. After half an hour, add 50 microliters of the provided stop solution to each well.
To stop the reaction, measure the absorbance at 490 nanometers to infect the pancreatic ASIN R cells with adenovirus after filtering the digested pancreatic tissue, but before flash freezing, the cells add 10 to the seven infectious units of adenovirus to the cell suspension and incubate the co-culture for 30 minutes at 37 degrees Celsius. Then evenly plate two milliliters of the cell suspension in each well of a six well plate. These images show as in our cells prepared for calcium measurements as visualized at 630 x magnification.
The cells were first visualized under brightfield microscopy. They were then loaded with the calcium dye flu oh four and visualized using fluorescence microscopy. Here an example of asar cell calcium measurements in response to physiologic stimuli is shown.
The asar cells were loaded with the calcium dye flu oh four and perfused with the acetylcholine analog carpool. The cells responded in the form of a calcium wave that initiates in the apical region and propagates to the basolateral region. Representative tracings shown in this figure demonstrate the typical peak plateau pattern commonly observed with one micromolar caracol.
Conversely, using sub maximal doses of the cholecystokinin analog, Ian yields oscillatory calcium responses. In this figure, the concentration dependent increases in LDH leakage in the presence of cerulean carbahol or the bile acid Toro lithocholic acid three sulfate or TLCS can be observed. The concentrations necessary to induce maximal leakage of LDH are consistent with those required to induce intra ASIN R protease activation and pathologic calcium signaling, suggesting that these events may lead to injury.
In this experiment, the ASIN R cells were infected with a luciferase reporter, adenovirus, which is driven by the promoter for a downstream calcineurin effector nuclear factor of activated T-cell or nfa. These representative data validate the infection method, demonstrating concentration and time dependent increases in N fat luciferase activity in the presence of TLCS Once mastered. This technique can be done in four to six hours depending on the conditions being tested while attempting this procedure.
It's important to be meticulous. This is particularly true during the cell preparation step of isolating the primary aser cells. After watching this video, you should have a good understanding of how to isolate primary as iner cells for the purpose of measuring cytosolic calcium levels and cell injury.
In addition, you should be able to reliably infect these cells with adenovirus.
