|
|
Blood, Vol. 111, Issue 4, 1924-1932, February 15, 2008
Differential context-dependent effects of friend of GATA-1 (FOG-1) on mast-cell development and differentiation
Blood Sugiyama et al.
111: 1924
Supplemental materials for: Sugiyama et al
Supplemental materials and methods Histamine release assay
For immunological activation, the mast cells were suspended in HEPES-Tyrode’s buffer (-) 10 mM HEPES, 130 mM NaCl, 5 mM KCl, 5.6 mM glucose and 0.1% BSA (pH 7.4), and sensitized with purified mouse anti-DNP IgE antibody (2 µg/mL, SPE-7, Sigma) 2 hours at 37°C. The supernatants were then removed by centrifugation and cells (2–3 × 104 per sample) were washed with HEPES-Tyrode’s buffer (-) and suspended in warm HEPES-Tyrode’s buffer (-). Next, the IgE-sensitized cells were challenged with DNP-HSA (0-1 µg/mL, Sigma) for 30 minutes at 37°C. Negative controls were treated in the same way, except for omission of IgE antibody. After stimulation, the incubation mixtures were cooled down to at 4°C and centrifuged to sediment the mast cells. The supernatants were collected and the mast cells were lysed in 1% NP 40. Histamine levels in dialysate fractions were determined by an HPLC-fluorometric method1 with slight modification.2 Histamine release (%) was calculated as : histamine in medium/(histamine in cells + histamine in medium) × 100. Phagocytosis analysis
GR-1+ cells sorted by FACSAria were seeded on to poly-D-lysine coated dish and incubated with Fluoresbrite™ Carboxylate 0.75 Micron Microspheres (polysciences, Inc., Warrington, PA) for 30 minutes at 37°C in a humidified atmosphere containing 5% CO2. Thereafter the cells were washed twice in medium. Microscopic images were obtained and captured using an Olympus IX70 microscope with an Uplan APO 20×/0.70 numeric aperture objective lens, an Olympus DP50 camera, and Viewfinder Lite software (all from Olympus). Images were processed using Adobe Photoshop (Adobe Systems).
Files in this Data Supplement:
- Figure S1 (JPG, 85.5 KB)
-
(A) Degranulation of ES cell-derived mast cells after crosslink of IgE receptors. Cells were sensitized by pre-incubation with mouse anti-DNP IgE antibody. The sensitized cells were then incubated for 2 hours with DNP-HSA after which histamine in the incubation medium and in the cells was measured. May-Giemsa staining of the control and stimulated cells are shown. (B) Histamine release from ES cell-derived mast cells after crosslink of IgE receptors. Histamine release was expressed as % of total cellular histamine released into the medium (% total). Data were the means derived from three samples and error bars indicated standard error of the mean (SEM). Statistically significant data compared with control (P<0.05 by Student’s paired t-test) was shown as asterisk.
- Figure S2. Latex phagocytosis analysis of the “neutrophils” obtained by the over-expression of FOG-1 (JPG, 82 KB)
-
After over-expression of FOG-1, GR-1+ cells were sorted and incubated with the FITC-conjugated microsphere at 37°C for 30 minutes.
REFERENCES
1. Yamatodani A, Fukuda H, Wada H, Iwaeda T, Watanabe T. High-performance liquid chromatographic determination of plasma and brain histamine without previous purification of biological samples: cation-exchange chromatography coupled with post-column derivatization fluorometry. J Chromatogr. 1985;344:115-123.
2. Mochizuki T, Yamatodani A, Okakura K, Takemura M, Inagaki N, Wada H. In vivo release of neuronal histamine in the hypothalamus of rats measured by microdialysis. Naunyn Schmiedebergs Arch Pharmacol. 1991;343:190-195.
|
|
