Immunofluorescence microscopy was used to examine the intracellular distribution of granules, cytoskeletons, or the localization proteins in RBL-2H3 cells. Cells grown on coverglass were fixed with 4% (wt/v) paraformaldehyde (PFA) at room temperature (RT) for 30 min, then permeabilized with 0.2% (v/v) Triton-X100 for 15 min. Cells were blocked with 1% bovine serum albumin (BSA) dissolved in PBS, then incubated with primary antibodies for 2 h at room temperature. Cells were washed 5 times with PBS. Alexa Fluor-conjugated secondary antibodies diluted 1:1000 were used as indicated. Oregon green 488 or Alexa 546 conjugated phalloidin diluted 1:2000 was used to stain F-actin and DAPI (4′, 6-diamidino-2-phenylindol) was used to stain nuclei. Cells were mounted on glass slides with ProLong™ Gold Antifade mounting media (ThermoFisher, Waltham, MA, USA). Images were captured by a Zeiss Observer Z1 microscope (Carl Zeiss, Oberkochen, Germany) with a 63X objective (1.4 NA) and processed using Axiovision 4.8 software.
Live-cell imaging was used to visualize the dynamics of granule trafficking by fluorescence microscopy using Lysotracker Red (ThermoFisher, Waltham, MA, USA) cell morphology transitions in a bright-field [11 (link)] and F-actin remodeling F-actin by LifeAct-mRuby [26 (link)]. Briefly, previously manipulated RBL-2H3 cells (e.g., Lifeact-mRuby transfected, GEF-H1-depleted or sensitized with anti-DNP-IgE) were grown on round coverslips. Coverslips were placed in an Attofluor chamber (ThermoFisher, Waltham, MA, USA) and growth media was replaced with HTB and placed on a 37 °C-heated microscope stage and objective. Images were captured using a PerkinElmer Ultra-VIEW VoX spinning disk confocal microscope (Waltham, USA) with a 63X objective (1.4 NA) using a 10 s imaging interval. After 1 min of imaging, resting cells were stimulated by the addition of 25 ng/mL of DNP-BSA, and drugs or DMSO were added at the same time. Volocity 6.0 software was used to record and analyze the live-cell videos, which were exported as Window Media files at 10 frames/s.
Live-cell imaging was used to visualize the dynamics of granule trafficking by fluorescence microscopy using Lysotracker Red (ThermoFisher, Waltham, MA, USA) cell morphology transitions in a bright-field [11 (link)] and F-actin remodeling F-actin by LifeAct-mRuby [26 (link)]. Briefly, previously manipulated RBL-2H3 cells (e.g., Lifeact-mRuby transfected, GEF-H1-depleted or sensitized with anti-DNP-IgE) were grown on round coverslips. Coverslips were placed in an Attofluor chamber (ThermoFisher, Waltham, MA, USA) and growth media was replaced with HTB and placed on a 37 °C-heated microscope stage and objective. Images were captured using a PerkinElmer Ultra-VIEW VoX spinning disk confocal microscope (Waltham, USA) with a 63X objective (1.4 NA) using a 10 s imaging interval. After 1 min of imaging, resting cells were stimulated by the addition of 25 ng/mL of DNP-BSA, and drugs or DMSO were added at the same time. Volocity 6.0 software was used to record and analyze the live-cell videos, which were exported as Window Media files at 10 frames/s.
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