4get BALB/c and IgE-deficient 4getxRag2−/−
mice have been described (Cheng et al., 2010
; Mohrs et al., 2001
). Mast cell-deficient Wsh
(Sash) mice on a C57BL/6 background were provided by G. Caughey (UCSF). MCPT5 cre mice were bred to either of the following reporter mice: Rosa-YFP, Ai6, or Rosa-DTA (Madisen et al., 2010
; Srinivas et al., 2001
; Voehringer et al., 2008
). Rosa-YFP and Ai6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in Specific Pathogen Free facilities. Experimental mice were 6–10 weeks old. Animal use was governed by and in accordance with approved protocols overseen by the Laboratory Animal Resource Center (LARC) and Institutional Animal Care and Use Committee (IACUC) at UCSF.
Skin tissue and peritoneal mast cell isolation
Ear tissue from euthanized mice was split into dorsal and ventral halves, and then minced. Tissue was re-suspended in PBS plus 2 U/ml of Liberase CI (Roche) and incubated at 37°C for 45 minutes in an orbital shaker similar to published protocols (Grimbaldeston et al., 2007
). Collagenase activity was quenched with PBS supplemented with 2% fetal calf serum (FCS), and the cells analyzed by flow cytometry. Peritoneal mast cells were analyzed from lavage fluid collected after euthanasia. The gating scheme was similar to a previously published study (Gessner et al., 2005
Construction of RFP-Fcε
RFP-Fcε was constructed and produced in the same manner as previous molecules (Cheng et al., 2010
). A primer pair (5’- TTTAGATCTGTGAGCAAGGGCGAGG-3’ and 5’-TGGATCCCTTGTACAGCTCGTCCATGC-3’) that spanned amino acids 2–476 was used to amplify the tdRFP cDNA (Shaner et al., 2004
Antibodies and flow cytometry
For mast cell staining, we used the following antibody clones: c-kit (ACK2, eBioscience, San Diego, CA), FcεRI (Mar-1, eBioscience). For basophil staining, we used: CD49b (DX5, eBioscience). For in vivo infusion, the following antibodies and clones were used: mouse IgE C38-2 (BD Pharmingen, San Diego, CA) anti-CD31 APC (390, eBioscience), tomato lectin FITC (Vector Labs, Burlingame, CA), anti-c-kit PE (2B8, eBioscience). For flow cytometry, we used an LSRII (Becton-Dickinson, San Jose, CA) for data acquisition and analysis. We performed post-acquisition analysis using FlowJo software (Treestar, Ashland, OR).
Mast cell blood sampling
One µg of PE-conjugated 2B8 or isotype control rat IgG2b antibody were infused I.V. into the tail veins of mice. Within five minutes, mice were euthanized with immediate processing of tissue for flow cytometry. Statistical analysis was performed using an unpaired t test.
For confocal microscopy, ear tissue from euthanized mice was split into dorsal and ventral halves (except for experiments involving bead infusions for which the ears remained intact). The tissue was then bathed in Vectashield (Vector Labs) and analyzed with a Nikon C1si laser scanning confocal microscope. Images were analyzed and rendered using Imaris software (Bitplane, Zurich, Switzerland). To analyze the distance from mast cells to blood vessels, we used Imaris software to mark the boundaries of the blood vessels and to generate “spots” to represent index populations. The software then calculated the distance from the center of each of these spots to the edge of the nearest blood vessel. A students t-test was performed for significance on the resulting data sets.
Mice received intraperitoneal injections of ketamine and xylazine for anesthesia. Mice were then placed in a lateral decubitus position on the imaging stage of a Nikon C1si microscope. The ventral half of the ear was then dissected away with preservation of blood flow to the dorsal half verified under light microscopy. The mouse was then secured on the stage with the microscope objective directed toward the dermal surface. Images were compiled, analyzed, and rendered using Imaris software (Bitplane, Zurich, Switzerland).
Monoclonal, anti-trinitrophenol IgE (C38-2) was biotinylated with EZ-Link Sulfo NHS (Thermo Scientific, Rockford, IL). After purification (Zeba Spin Desalting columns, Thermo Scientific), 40 µg of IgE (4B12, Vector Labs) was then combined with ~109 Dynal MyOne T1 streptavidin beads for 15 minutes (Invitrogen, Carlsbad, CA). To quench unoccupied streptavidin moieties and label the bead, 40 µg of dextran (3 kDa) coupled to biotin and tetramethylrhodamine was added to the mixture for 15 minutes (Invitrogen). The beads were then washed and resuspended in PBS prior to I.V. infusion. Ten minutes after infusion, ears were dissected from euthanized mice and analyzed intact using confocal microscopy. For the bead recovery assay, ears were harvested 20 minutes after infusion, weighed, and placed directly into digest buffer (0.25% SDS, 0.1 M NaCl, 50mM Tris pH 8, 7.5 mM EDTA) with an additional 0.5 mg/ml Proteinase K for 3 hours at 56 degrees and constant agitation. Magnetic beads were then isolated using a Dynal magnet (Invitrogen, Carlsbad, CA) after multiple washes with PBS and resuspended in 100 ul of water. The beads were then counted manually with a hemacytometer.
Four hours after I.V. IgE infusion or IP challenge with 1 mg of TNP-ovalbumin in sensitized animals, peritoneal lavage fluid was isolated and ckit+GFP+ mast cells were stained with anti-CD107a (1D4B, eBioscience).
Histamine blockade and cromolyn sodium administration
Mice received inhibitors of mast cell degranulation (cromolyn) and histamine blockade per published protocols (Dawicki et al., 2010