Isolation and Culture of Bone Marrow-derived MCs

Femoral and tibial bones from the hind leg of C57BL/6 male mice were used for isolation of bone-marrow cells. The cells were then cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 25 mM glucose and pyruvate, supplemented with 10% heat-inactivated fetal bovine serum, 60 µg/ml penicillin, 50 µg/ml streptomycin sulphate, 2 mM L-glutamine and 20 ng/ml recombinant mouse IL-3. The cells were maintained at 0.5–1 × 106 cells/ml by weekly changes of medium. From day 10, 50% of the cells were cultured in supplemented DMEM media as above but containing only 5 mM glucose. The obtained BMMCs, maintained either in media with 5 mM glucose or 25 mM glucose, were used in experiments.

Viability of MCs

Cell viability was monitored using the PrestoBlue reagent (C.N. #A13261; Invitrogen, CA) or with trypan blue (#T10282, ThermoFisher Scientific, Watham, MA); cells were mixed with trypan blue at a 1:1 ratio, and cell viability was determined with Countess II FL (ThermoFisher Scientific, Watham, MA).

Trypsin-like Activity

Ten µl of cell supernatants were mixed with 90 µl H2O and 20 µl (1.8 mM) of the chromogenic substrate S-2288 (Instrumentation Laboratory SpA-V, Milano, Italy) in 96-well plates (#82.1581, Sarstedt, Nümbrecht, Germany). Absorbance at 405 nm was monitored.

Stimulation of Cells

Bone marrow-derived MCs (BMMCs) (1 × 106 cells/ml) were sensitized overnight with IgE anti-DNP (Merck, # D8406-100 µg, Darmstadt, Germany) at 0.1 μg/ml. Next, cells were washed twice with Tyrode's buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES, and 0.1% BSA, pH 7.4), suspended at 2 × 106 cells/ml either in Tyrode’s buffer (for β-hexosaminidase activity, qPCR analysis) or in cell culture medium (for ELISA) and pipetted into 48-well plates (#83.3923, Sarstedt, Nümbrecht, Germany) at 1 × 106 cells/well. The cells were treated with inhibitors of GLUT1 (Bay876; Merck, # SML1774-5mg, Darmstadt, Germany) or GLUT3 (G3iA; ChemDiv, #D430-1191, San Diego, CA) for 1 h, followed by activation by adding DNP-HSA (0.5 μg/ml) (Merck, #A6661-100mg, Darmstadt, Germany). For β-hexosaminidase activity and mRNA expression, MCs were activated for 1 h. For ELISA measurements, activation with DNP-HSA was performed for 4 h. For stimulation with C48/80 (Merck, #C2313, Darmstadt, Germany), the cells were suspended either in Tyrode’s buffer or in cell culture medium (2 × 106 cells/ml), pipetted into 48-well plates (1 × 106 cells/well), and pretreated with GLUT1 or/and GLUT3 inhibitor. After 1 h, cells were activated with 50 µM C48/80 for 60 min (for β-hexosaminidase activity and qPCR analysis) or 4 h (ELISA).

Peritoneal Cell-derived MCs (PCMCs)

Abdominal cavities of C57BL/6 male mice were rinsed with 7 ml Tyrode`s buffer (130 mM NaCl, 5 mM KCl, 1.4 mM CaCl2, 1 mM MgCl2, 5.6 mM glucose, 10 mM HEPES and 0.1% BSA; pH 7.4). Collected cells were rinsed twice with Tyrode’s buffer and once with Dulbecco's Modified Eagle Medium (DMEM) containing glutamax and pyruvate, supplemented with 10% heat-inactivated fetal bovine serum, 60 µg/ml penicillin, 50 µg/ml streptomycin sulphate, 1 mM non-essential amino acids, 50 µM β-mercaptoethanol, 20 ng/ml recombinant mouse IL-3 and 20 ng/ml recombinant mouse stem cell factor (SCF). Peritoneal cells from individual mice were cultured in medium as above at 0.5 × 106 cells/ml. After three days, all cells were pooled. On day 16, the cells were analyzed for the expression of c-kit (anti c-kit-FITC: Invitrogen, ref.# 11-1171-82) and FcɛR1 (anti FcɛR1-PE: eBiosciences, ref.# 12-5898-82) using a BD Accuri C6 Plus flow cytometer (BD Biosciences). Cytospin slides of cells were stained with toluidine blue and pictures were taken with a Nikon 90i microscope (Nikon Instruments Europe, Amsterdam, Netherlands). Mature PCMCs were split into two cultures, one maintained in 5 mM glucose and the other in 25 mM glucose.

Glucose Treatment

Bone marrow-derived MCs (BMMCs) were cultured in complete DMEM media with 5 mM glucose. When studying the effect of glucose on cell morphology and degranulation of BMMCs, the glucose concentration was adjusted to 12.5 mM, 15 mM and 25 mM with 2 M glucose (VWR, ref.# 101174Y) prepared in H2O.

ELISA

Cytokine levels in media from treated cells was determined by ELISA kits: mouse IL-6 (ThermoFisher Scientific; # 88-7064) and mouse TNFα (Thermo Fischer; #88-7324).

Cell Morphology and Degranulation

To examine morphological effects of glucose on MCs, cytospin slides were prepared using approximately 0.5 × 104 cells/slide. Cells were stained with toluidine blue. MC degranulation (∼2 × 106 cells in Tyrode’s buffer) was quantified by measuring β-hexosaminidase release [25].

Immunofluorescence and Confocal Microscopy

For immunofluorescence analysis, cells were first settled onto glass slides and fixed with cold acetone (-20 °C) for 15 min. Staining with antibodies was performed following the protocol described [26]. Briefly, slides were first blocked with normal horse and or goat serum, followed by rinsing with PBS (3×) and incubation overnight (at 4 °C, in dark) with the primary antibody: rabbit monoclonal to GLUT1 (1:100; C.N #ab115730, Abcam, Amsterdam, Netherlands), or rabbit polyclonal to GLUT3 (1:100; C.N #ab234756, Abcam, Amsterdam, Netherlands). All primary antibodies were diluted with PBS/1% BSA/0.3% Triton-X100 to permeabilize the cells. Next, cells were washed in PBS (3 × 5 min), and then incubated for 60 min (on a shaker) at room temperature with biotinylated secondary antibody. As secondary antibody, biotinylated goat anti-rabbit IgG (#BA1000) or biotinylated horse anti-rabbit IgG (#BA1100, Vector Laboratories, Burlingame, CA; 1:250 dilution in PBS/1% BSA) was used. Finally, slides were incubated with streptavidin-Cy2 (for GLUT1; 1:2,000, Amersham Int., Poole, UK) or streptavidin-Cy3 (for GLUT3; 1:5,000, Amersham Int. Poole, UK). For visualization of nuclei, DAPI (4,6-diamidino- 2-phenylindole, Invitrogen, CA) staining was performed (1:500 diluted in PBS). Digital images were captured by a confocal microscope (Zeiss LSM700, Carl Zeiss, Berlin, Germany) and analyzed with ZEN 2009 software (LSM 710; Carl Zeiss, Berlin, Germany). All photographs were taken at original magnifications of 200×, 400× or 630× with oil objective.

RNA Isolation and qPCR

Total RNA was isolated using the NucleoSpin® RNA isolation kit (MACHEREY-NAGEL, Düren, Germany). RNA purity and concentration was measured by a NanoDrop™ 2000 Spectrophotometer (Thermo Scientific™, Wilmington, DE) and the ND-1000 V3.7.0 program. First-strand cDNA was synthesized using 200 ng of total RNA as template and the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA), on a SimpliAmp Thermal Cycler instrument (Applied Biosystems by Life Technologies/Thermo Fisher Scientific, Darmstadt, Germany). Subsequently, qPCR was performed using up to 100 ng of cDNA, 400 nM primers (indicated below) and iTaq Universal SYBR Green Supermix (Bio-Rad, Hercules, CA), following the PCR cycling conditions recommended by the manufacturer (on a C1000 Touch Thermal Cycler instrument (Bio-Rad, Hercules, CA). Each sample was analyzed in duplicates, and qPCR data was analyzed by using the Bio-Rad CFX Maestro program. The primer efficiency of each primer pair was checked by the iTaqTM Universal SYBR® Green (BioRad, Hercules, CA) Supermix protocol. When a satisfactory primer efficiency (range between 80–110%) and dissociation curve (slope -3.1 to -3.6) was obtained, a qPCR reaction was run in 384-well microtiter plates (Sarstedt, Nümbrecht, Germany) with 5 min centrifugation (2,000 × g) prior to qPCR (CFX384 Touch™, BioRad). The cycling conditions were as follows: Step 1: 95 °C (10 min); step 2: 95 °C (15 s); step 3: 60 °C (60 s); step 4: 72 °C (20 s). Repeat steps 2–4, 40× + dissociation stage (BioRad).

Primer sequences were as follows: GLUT1/Slc2A1, forward: 5′-TCAACACGGCCTTCACTG-3′; GLUT1/Slc2A1, reverse: 5′-CACGATGCTCAGATAGGACATC-3′; GLUT3/Slc2A3, forward: 5′-ATGGGGACAACGAAGGTGAC-3′; GLUT3/Slc2A3, reverse: 5′-CAAAGCTATCACGGAGATGACG-3′; IL-6 forward: 5´-AGACAAAGCCAGAGTCCTTCAGAGA-3´; IL-6, reverse: 5´-TAGCCACTCCTTCTGTGACTCCAGC-3´; TNFα, forward: 5′-CCACATCTCCCAGAAAA-3´; TNFα, reverse: 5′-AGGGTCTGGGCCATAGAACT-3′; Ccl4, forward: 5′- TTCCTGCTGTTTCTCTTACACCT-3´; Ccl4, reverse: 5′- CTGTCTGCCTCTTTTGGTCAG-3′; Gapdh, forward: 5′-CTCCCA CTCTTCCACCTTCG-3´; Gapdh, reverse: 5′-CCACCACCCTGTTGCTGTAG-3′.

AmpliSeq Transcriptome Analysis

Total RNA was isolated using NucleoSpin® RNA II (Macherey Nagel, 740955) and was used for AmpliSeq transcriptome analysis as described [27]. The displayed data were derived from previous studies [28,29,30].

Statistical Analysis

All data were analyzed with one or two-way analysis of variance (Anova) followed by Tukey's or Dunnett`s multiple comparisons test. The results were analyzed using GraphPad Prism 8.1.0 (GraphPad Software, CA). p-values ≤ 0.05 were considered to represent statistical significance. The presented data are either the average of two independent experiments or from individual experiments, representative of at least 2 independent experiments.