Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca²⁺-imaging and Degranulation Assays

All animal procedures were performed according to the German legislation guidelines for care and use of laboratory animals (officially approved by the Karlsruhe regional council).

1. PMC Isolation and Cultivation by Intraperitoneal Lavage

Table 1: Materials for Step 1.

1. PMC isolation by intraperitoneal lavage
   1. Prior to the cell isolation, prepare the materials listed in Table 1.
   2. Use 8 to 14 week-old male mice for PMC isolation. Euthanize a mouse by CO₂ inhalation, and confirm the death by loss of reflexes. Spray the mouse with 70% ethanol and fix it on a foam block using pins (dorsal side down). Remove the ventral skin of the mouse using blunt edge scissors. Avoid damaging the peritoneal cavity.
      NOTE: We use typically this protocol for mice with a C57BL/6N strain genetic background.
   3. Inject 7 mL of the ice-cold RPMI medium and 5 ml of air in the peritoneal cavity using a 10 mL syringe equipped with a 27 G needle. Push the needle carefully in the peritoneum and do not perforate any organs. Use a spot in the region of the epididymal fat to reduce the risk of an organ perforation.
   4. After injection, shake the mouse for 1 min to detach peritoneal cells into the RPMI medium. Do not shake the mouse too strongly, to avoid damaging the internal organs and contaminating the peritoneal cavity with the blood.
   5. Reuse the 10-mL syringe by equipping it with a new 20 G 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. Insert a 20 G needle, bevel up, and aspirate the fluid from the abdomen gently and slowly (~0.5 mL/s) to avoid clogging by the inner organs. Collect as much fluid as possible (typically 5–6 mL).
   6. 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.
   7. Centrifuge the tubes with the cell suspension at ~300 x g for 5 min. Under a sterile hood aspirate the supernatant. For each mouse, combine the sample pellets in 4 mL of cold (4–10 °C) PMC Medium and transfer the cell suspension to a 25 cm² culture flask. Add the growth factors IL-3 and SCF to the final concentrations 10 ng/mL and 30 ng/mL, respectively. Place the flask containing the cells in an incubator (37 °C and 5% CO₂) and incubate for ~48 h until the next procedure.
2. PMC culture
   1. Day 2 (~48 h after isolation): Medium change
      1. To remove the supernatant and non-adherent cells (erythrocytes and dead cells), aspirate the medium from the flask by placing a Pasteur pipette tip at the edge of the flask and holding the flask almost horizontally. Add 4 mL of pre-warmed PMC Medium per mouse. Add the growth factors IL-3 (10 ng/mL) and SCF (30 ng/mL). Place the flask containing the cells in an incubator (37 °C and 5% CO₂).
   2. Day 9: Cell splitting
      1. Transfer the cell suspension from the cell culture flask into a 50 mL plastic centrifuge tube. Wash the flask three times with 10 mL pre-warmed (37 °C) Dulbecco's phosphate-buffered saline (DPBS), collecting the cell suspension with the detached cells in the same 50 mL plastic centrifuge tube. Count the cell concentration by a hemocytometer, and calculate the total number of the cells.
      2. Centrifuge the cell suspension at ~300 x g for 5 min and aspirate the supernatant. Recover the pellet in pre-warmed PMC Medium (37 °C) to obtain the cell concentration 1 x 10⁶ cells/mL. Transfer the cell suspension to a new 25 cm² culture flask. Discard the old flask with fibroblasts adhering to the bottom.
      3. Add the growth factors IL-3 (10 ng/mL) and SCF (30 ng/mL), and place the flask containing the cells in an incubator (37 °C and 5% CO₂).
   3. Days 12–15: Cell measurements
      1. If any experiments with stimulation of FcɛRI receptors are planned, pretreat the cells overnight with 300 ng/mL of IgE anti-DNP in standard culture medium. Proceed with step 2 or step 3.

2. Fluorometric Intracellular Free Calcium Concentration Measurement in PMCs

1. Prior to the measurements, prepare the materials listed in Table 2. Also, prepare an Imaging Setup consisting of: Inverted Fluorescence Microscope; Objective: 40X/1.3 Oil; Fura 2 Filter Set; CCD Camera; Excitation Light Source; Imaging Acquisition Program; Gravity fed solution application system.

Table 2: Materials for Step 2.

2. Fura-2 imaging preparation
   1. Perform a cell count using a hemocytometer and adjust the cell concentration to 1 x 10⁶ cells/mL. Split the PMCs colonies by gently pipetting (3–5 times) the cell suspension with a 1 mL pipette tip.
   2. Prepare a Ca²⁺-containing HEPES buffered salt solution (Ca²⁺-HBSS) composed of 135 mM NaCl, 6 mM KCl, 2 mM CaCl₂, 1.2 mM MgCl₂, 10 mM HEPES, and 12 mM glucose. Adjust the pH to 7.4 with 3 M NaOH.
   3. Prepare concanavalin A coated slides by pipetting 1 drop of concanavalin A solution (0.1 mg/mL in H₂O) on each 25 mm round cover glass under a sterile hood. Leave the drops on the cover slips for at least 1 min, then remove most of the solution with a pipette and let the cover slips air-dry for 45 min. Carefully press the concanavalin A treated cover slips on the rubber imaging chamber (see Table of Materials) until they attach to obtain microscopy imaging chambers.
      NOTE: Poly-L-Lysine coated slides can be used as an alternative.
   4. Dissolve the fluorescent Ca²⁺ dye Fura-2 AM (1 mM) in dimethyl sulfoxide (DMSO). Store this solution protected from light.
   5. Dilute the Fura-2 AM stock solution in Ca²⁺-HBSS to a final concentration of 5 µM in order to prepare the "loading buffer". Supplement with 5 µL/mL of 20% pluronic F-127 in DMSO.
   6. Mix 500 µL of the "loading buffer" with 500 µL of the PMC cell suspension. Pipette the mixture into the imaging chamber and incubate the cells for 20–30 min at room temperature in the dark.
   7. Set up the gravity fed application system with different solutions according to the stimulation protocol. Supplement syringe 1 with 40 mL of Ca²⁺-HBSS solution, syringe 2 with 40 mL of Ca²⁺-HBSS solution containing 100 ng/mL DNP, syringe 3 with 40 mL of Ca²⁺-HBSS solution containing 50 µg/mL Compound 48/80, syringe 4 with 40 mL of nominally Ca²⁺-free-HBSS solution, and syringe 5 with 40 mL of nominally Ca²⁺-free-HBSS solution containing 2 µM Thapsigargin.
   8. Prepare a 40X high-aperture oil immersion objective by placing a small drop of immersion oil on it.
   9. Mount the application system on the stage of the inverted microscope. Put the imaging chamber with the loaded cells in the application system and secure it to prevent movement during recording. Turn on the transmitted light and focus the cells.
   10. Rinse the cells three times with Ca²⁺-HBSS buffer using the gravity-fed application system.
   11. Incubate the cells in Ca²⁺-HBSS for 5 min for Fura-2 AM de-esterification.
   12. Rinse the cells one more time before starting the imaging, to remove any potentially leaked fluorescent dye.
3. Cell imaging
   1. Start the imaging acquisition program in the physiological acquisition mode. Open the setup window, and adjust the focus and the optimal acquisition time (which should be the same for excitation with 340 nm and 380 nm and typically ranges between 50–150 ms).
      NOTE: Minimize the exposure of the Fura-2 loaded cell to any light to avoid photobleaching of the dye.
   2. Image a reference picture, and mark the cells as regions of interest (ROI). Mark the last ROI in an area where no cells are present and define it as the background.
   3. Complete the setup and start the measurement with a 5 s acquisition rate.
      NOTE: The cells will be alternately illuminated with the excitation light of 340 nm and 380 nm, and the emitted fluorescence (>510 nm) will be collected using the CCD camera. The images of fluorescence signals F340 nm and F380 nm as well as the ratio (F340 nm/F380 nm) will be displayed in three windows. The charts of time course of individual ROIs fluorescence intensity mean values will be also continuously displayed.
   4. Add solutions at the proper cycle number according the design of the experiment:
      1. To measure antigen-induced elevation of [Ca²⁺]_i, as illustrated in Figure 2A, 2B, apply the syringe 2 solution after cycle 20, and stop recording after cycle 150.
      2. To measure Compound 48/80-induced elevation of [Ca²⁺]_i,, as illustrated in Figure 2C, apply the syringe 3 solution after cycle 20, and stop recording after cycle 150.
      3. To measure the elevation of [Ca²⁺]_i evoked by SOCE, as illustrated in Figure 2D, apply the syringe 4 solution after cycle 10, apply the syringe 5 solution after cycle 70, apply the syringe 4 solution after cycle 120, apply the syringe 1 solution after cycle 150, and stop recording after cycle 220.
   5. After finishing the measurement, convert the results into a ratio table containing the measured intensities for both excitation wavelengths with and without background correction, as well as the ratio values with and without background correction for every ROI and every measurement time point. In the acquisition program, consecutively press the buttons: "start cutter", "mark all", "convert all", "physiology measurements", "Ok, "Ok". Save the files as tables and image stacks for off-line analysis.

3. Beta-hexosaminidase Release Measurement in PMCs

Table 3: Materials for Step 3.

1. Prior to the measurements, prepare the materials listed in Table 3.
   NOTE: β-hexosaminidase released from MCs after their stimulation hydrolyzes p-nitrophenyl-acetyl-D-glucosamine (pNAG) into p-nitrophenol and N-acetyl-D-glucosamine. The amount of β-hexosaminidase in the sample is proportional to the amount of p-nitrophenol that will be formed. In a high pH environment of "Stop Solution", p-nitrophenol exists as fully deprotonated p-nitrophenolat and can be detected by its light absorbance at 405 nm.
2. Prepare Tyrode's solution containing 130 mM NaCl, 5 mM KCl, 1.4 mM CaCl₂, 1 mM MgCl₂, 10 mM HEPES, 5.6 mM glucose, and BSA 0.1%. Adjust the pH to 7.4 with 3 M NaOH.
3. Prepare the lysis solution by adding a 1% volume of Triton X-100 to the Tyrode's solution.
4. Prepare the stop solution composed of 200 mM glycine and adjust the pH to 10.7 with NaOH.
5. Prepare the pNAG solution (4-Nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside) solution by dissolving pNAG in 0.4 M citric acid to a final concentration of 4 mM.
6. Calculate the amount of the cells needed for the assay (perform the assay in duplicate). Use 200,000 PMCs per well. Use 2 wells as negative controls (Tyrode's solution without any secretagogues such as DNP or compound 48/80) and 2 wells as positive controls (Tyrode's solution with 10 µM Ionomycin) for every genotype.
7. Transfer the cells into a 15-mL plastic centrifuge tube, adjust the volume to 15 mL with Tyrode's solution, and centrifuge at 200 x g for 4 min. Remove the supernatant with a Pasteur pipette and resuspend the cells in Tyrode's solution to 2 x 10⁶ cells/mL.
8. Transfer 100 µL of the cell suspension per well to a 96-well V-bottom well plate. Add 25 µL of either stimulation or control solution to each well (according to the pipetting scheme illustrated in Figure 3A). Incubate the cells for 45 min at 37 °C and 5% CO₂.
9. Stop the reaction by placing the 96-well plate on ice for 5 min. Then, centrifuge the plate at 4 °C for 4 min at 120 x g. Transfer 120 µL of the supernatant using a multichannel pipette to a flat-bottom 96-well plate and place it on ice. Carefully avoid touching the cell pellets, but completely aspirate the supernatant.
10. Add 125 µL of lysis buffer to the cell pellets. Incubate the cell pellets for 5 min at room temperature and resuspend them after the incubation by repeated pipetting (approximately 5 times).
11. Pipette 25 µL of the pNAG solution (4 mM) in the required wells of a new flat-bottom 96-well plate. Add 25 µL from each supernatant to the prepared pNAG solution as well as 25 µL of each cell lysate.
12. Incubate the reaction batches for 1 h at 37 °C. After the incubation, pipette 150 µL of stop solution to each well to stop the reaction.
13. Analyze the plate with a microplate reader using a dual-wavelength setting at 405 nm with reference 630 nm for automatic background subtraction. In the acquisition program, tick the box "Reference" and in the "Absorbance" program element menu, select "630 nm" from the drop-down list. If the optical density of the samples is too high, prepare an appropriate dilution of the probe before remeasuring.
14. Calculate the percentage of β-hexosaminidase release according to the following equation:
    
    where Release [%] is the percent of specific beta-hexosaminidase release, A[supernatant] is the background corrected absorption of the supernatant, and A[lysate] is the background corrected absorption of the corresponding cell lysate.