Source: Tsvilovskyy, V., et al. Isolation of Peritoneum-derived Mast Cells and Their Functional Characterization with Ca2+-imaging and Degranulation Assays. J. Vis. Exp. (2018).
This video demonstrates a colorimetric assay to determine the degranulation activity of mast cells. The enzyme released by the mast cells hydrolyzes the substrate and produces a colored product. The degranulation activity of the mast cells was measured as the percentage release of enzymes outside the cell normalized with the total enzyme content in the cell.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. PMC Isolation and Cultivation by Intraperitoneal Lavage
2. Beta-hexosaminidase Release Measurement in PMCs
Table 1: Materials for Step 1.
1 | Ice |
2 | 10 mL syringes |
3 | 27 G needles |
4 | 20 G needles |
5 | Styrofoam block and pins |
6 | Collection tubes (50 mL Plastic Centrifuge Tubes) |
7 | 70% ethanol |
8 | RPMI Medium (Pre chilled and kept on ice) |
9 | PMC Medium: RPMI Medium + 20% FCS (Fetal Calf Serum) + 1% Penicillin Streptomycin solution (Pen-Strep) |
10 | Dulbecco’s phosphate buffered saline – DPBS (without Ca2+ and Mg2+) |
11 | Culture Flasks (25 cm) |
12 | Growth factors stock solutions (IL-3: 1 μg/μL; SCF: 2.5 μg/mL) |
13 | Serological pipettes (10 mL) |
14 | Pipettes tips sterile (20–200 µL) |
15 | Hemocytometer |
16 | Bench Centrifuge |
17 | Scissors and forceps |
18 | CO2 chamber for mice |
19 | Open sterile hood |
20 | Closed sterile hood |
21 | Cell incubator (37 °C and 5% CO2) |
22 | Transfer pipettes (20–200 µL) |
Table 2: Materials for Step 2.
1 | PMC (12–15 days old) 1 x 106 cells/mL |
2 | Anti-DNP-Antibody (IgE) stock solution |
3 | DNP-HSA stock solution |
4 | Compound 48/80 stock solution |
5 | Ionomycin |
6 | 96-Well plate, v-shaped bottom |
7 | 96-Well plates, flat bottom |
8 | Plastic centrifuge tubes (15 mL) |
9 | Serological pipettes |
10 | Cell incubator (37 °C and 5% CO2) |
11 | Bench Centrifuge |
12 | Microtiter plate reader for optical density measurements |
13 | Multichannel pipette (20–200 μL) |
14 | Hemocytometer |
Figure 1: Beta-hexosaminidase release colorimetric multi-well measurements performed with PMCs isolated from wild-type mice. (A) Stimulation scheme of 96-well plate pipetting for performing the beta-hexosaminidase assay; results are presented in panel B. "Spontan" corresponds to pipetting of Tyrode's Solution without the addition of any secretagogues; IM = Ionomycin; Cp 48/80 = Compound 48/80. (B) Bar graph illustrating the percentage of ß-hexosaminidase release under basal conditions (Spont), after stimulation with Ionomycin (IM), dinitrophenol-human serum albumin (DNP), and Compound 48/80 (Cp 48/80) with indicated concentrations. The assay was performed in duplicate; error bars show the standard error of the mean.
The authors have nothing to disclose.
RPMI Medium | Thermo Scientific | 21875034 | |
DPBS | Sigma-Aldrich | 14190144 | |
FCS | Thermo Scientific | R92157 | |
IL-3 | R&D Systems | 403-ML | |
SCF | Thermo Scientific | PMC2115 | |
DNP-HSA | Sigma-Aldrich | A6661 | |
Anti-DNP-Antibody | Sigma-Aldrich | D-8406 | |
Penstrep | Thermo Scientific | 15140122 | |
Compound 48/80 | Sigma-Aldrich | C2313 | |
4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol | Sigma-Aldrich | X100 | |
4-Nitrophenyl 2-acetamido-2-deoxy-β-D-glucopyranoside | Sigma-Aldrich | N9376 | |
CoverWell Imaging Chamber | Sigma-Aldrich | GBL635051 | |
96-Well plate, v-shaped bottom | Corning | 3896 | |
96-Well plates, flat bottom | Greiner Bio-One | 655180 | |
Microtiter plate reader with "i-control" software | Tecan | Nano Quant, infinite M200 Pro | |
Flat bottom plate | Greiner Bio-One | 655180 | |
Ionomycin | Sigma-Aldrich | I9657 | |
50 mL Plastic Centrifuge Tubes | Sarstedt | 62.547.254 | |
Culture Flasks (25 cm) | Greiner Bio-One | 69160 | |
Hemocytometer | VWR | 631-0696 | |
Monochromatic Light Source | Sutter Instruments | Lambda DG-4 | |
15 mL Plastic Centrifuge Tubes | Sarstedt | 6,25,54,502 | |
Bench Centrifuge | Heraeus | Megafuge 1.0R |