These methods can help answer key questions in the field of intracellular signaling on excitable cells, such as which ion channels are critical for immune cell activation and mediator release. The main advantages of these techniques are their relative simplicity and the possibility of simultaneous analysis of large number of cells. Demonstrating Fura-2 calcium imaging in mast cells will be Kathrin Ohlenschlager, a graduate student from my laboratory.
To begin this procedure, spray the euthanized mouse with 70%ethanol and fix it on a foam block with its dorsal side down using pins. Using blunt-edge scissors, remove the skin over the abdomen of the mouse and avoid damaging the peritoneal cavity. Next, inject seven milliliters of ice-cold RPMI medium in the peritoneal cavity by inserting a 27 gauge needle carefully in the peritoneum.
Do not perforate any organs, and use a spot in the region of the epididymal fat to reduce the risk of an organ perforation. After injection, shake the mouse for one minute to detach the peritoneal cells into the RMPI medium. Do not shake the mouse too strongly to avoid damaging the internal organs and contaminating the peritoneal cavity with the blood.
Then, reuse the syringe by equipping it with a new 20 gauge cannula. Shift the inner organs to one side by tilting the foam block and gently tapping it on the bench to make medium aspiration easier from the other side. Subsequently, insert a 20 gauge needle bevel-up to aspirate the fluid from the abdomen gently and slowly to avoid clogging by the inner organs.
Collect as much fluid as possible. Then, remove the needle from the syringe and transfer the collected cell suspension in a collection tube on ice. Discard a sample tube if there is visible blood contamination.
Centrifuge the tube with clean cell suspension at 300 g for five minutes. Under a sterile hood, aspirate the supernatant. Re-suspend the sample pellets in cold PMC medium and transfer the cell suspension to a 25-square-centimeter cell culture flask.
Subsequently, add the growth factors IL-3 and SCF to the final concentrations of 10 nanograms per milliliter and 30 nanograms per milliliter, respectively. Incubate the cells for approximately 48 hours at 37 degrees Celsius until the next procedure. On days 12 through 15, if any experiments with stimulation of plasmalemmal IgE receptors are planned, pretreat the cells overnight with 300 nanograms per milliliter of IgE anti-DNP in standard culture medium.
To prepare Fura-2 imaging, dilute Fura-2, AM stock solution in calcium HBSS to a final concentration of five micromolar in order to prepare the loading buffer. Then, mix 500 microliters of the loading buffer with 500 microliters of the PMC cell suspension. Pipette the mixture into the imaging chamber and incubate the cells for 20 to 30 minutes at room temperature in the dark.
Set up the gravity-fed application system with different solutions according to the stimulation protocol. Next, mount the application system on the stage of the inverted microscope. Prepare a 40X high-aperture oil immersion objective by placing a small drop of immersion oil on it.
Place the imaging chamber with the loaded cells in the application system and secure it to prevent movement during recording. Then, turn on the transmitted light and focus on the cells. Subsequently, start the imaging acquisition program in the Physiological Acquisition mode.
Open the setup window and adjust the focus and the optimal acquisition time. Image a reference picture and mark the cells as ROIs. Mark the last ROI in an area where no cells are present and define it as the background.
Then, complete the setup and start the measurement with a five-seconds-acquisition rate. Next, add solutions at the proper cycle number according to the design of the experiment. To measure antigen-induced elevation of intracellular calcium concentration, apply the syringe-two solution after cycle 20 and stop recording after cycle 150.
To measure compound 48/80-induced elevation of intracellular calcium concentration, apply the syringe-three solution after cycle 20 and stop recording after cycle 150. To measure the elevation of intracellular calcium concentration evoked by SOCE, apply the syringe-four solution after cycle 10, apply the syringe-five solution after cycle 70, apply the syringe-four solution after cycle 120, apply the syringe-one solution after cycle 150, and stop recording after cycle 220. In the acquisition program, consecutively press the buttons Start Cutter, Mark All, Convert All, Physiology Measurements, OK, and then OK again.
Save the files as tables and image stacks for offline analysis. To measure beta-hexosaminidase release, transfer 100 microliters of the cell suspension per well to a 96-well V-bottom well plate. Add 25 microliters of either stimulation or control solution to each well.
Then, incubate the cells for 45 minutes at 37 degrees Celsius. Afterward, stop the reaction by placing the 96-well plate on ice for five minutes. Then, centrifuge the plate at four degrees Celsius for four minutes at 120 g.
Transfer 120 microliters of the supernatant to a flat-bottom 96-well plate and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant. Next, add 125 microliters of lysis buffer to the cell pellets.
Incubate the cell pellets for five minutes at room temperature and re-suspend them after the incubation by repeated pipetting. Pipette 25 microliters of the four-millimolar pNAG solution in the required wells of a new flat-bottom 96-well plate. Add 25 microliters of each supernatant and 25 microliters of each cell lysate separately to the prepared pNAG solution.
Incubate the reaction batches for one hour at 37 degrees Celsius. After the incubation, pipette 150 microliters of stop solution to each well to stop the reaction. Next, analyze the plate sample light absorbance with a micro-plate reader at 405 nanometers with reference 630 nanometers for automatic background subtraction.
In the acquisition program, check the box Reference, and in the absorbance program element menu, select 630 nanometers for automatic background subtraction. Beta-hexosaminidase release assay was performed to test the ability of the isolated PMCs that undergo degranulation upon crosslinking of the plasmalemmal IgE high-affinity receptors or stimulation of the Mrg PRB2 receptors. The cells were added to a V-bottom 96-well plate and stimulated according to this scheme for 45 minutes at 37 degrees Celsius.
After centrifugation and removal of the supernatant, the pellets were lysed. The beta-hexosaminidase content of individual supernatants and pellet lysates was then analyzed by their reactions with pNAG during a one-hour incubation at 37 degrees Celsius. The amount of the reaction products was detected colorimetrically and the percentage of the released beta-hexosaminidase was calculated.
The spontaneous release was very low, whereas the responses to strong degranulation stimuli, such as 10 micromolar ionomycin and compound 48/80 at 50 micrograms per milliliter, were over 40%and 60%respectively. Degranulation responses to DNP stimulation were dose-dependent over the concentration range of 10 to 300 milligrams per milliliter. Together, these data clearly indicate a good functional state of the isolated PMCs.
Using this technique, you will obtain highly pure population of murine mature connective tissue mast cells derived from peritoneum within two weeks. While attempting this procedure, it's important to remember to maintain sterile conditions during the cell isolation and culturing to avoid bacterial contamination. Following this procedure, other methods, like electrophysiological patch-clamp ion current measurements can be performed in order to answer additional questions concerning calcium entry pathways in mast cells.
After its development, Fura-2 intracellular calcium imaging technique paved the way for researchers in the field of mass cell physiology to explore calcium dependence of mast cell degranulation. After watching this video, you should have a good understanding of how to perform Fura-2 calcium imaging experiments and beta-hexosaminidase degranulation assay with mast cells. Don't forget that working with UV radiation can be hazardous for eyes and skin, and unnecessary exposure should be avoided while performing this procedure.
