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.
Visualizing Intracellular Dynamics in RBL-2H3 Cells
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.
Corresponding Organization : University of Alberta
Variable analysis
- Cell manipulations (e.g., Lifeact-mRuby transfected, GEF-H1-depleted or sensitized with anti-DNP-IgE)
- Stimulation with 25 ng/mL of DNP-BSA
- Addition of drugs or DMSO
- Intracellular distribution of granules, cytoskeletons, or the localization of proteins in RBL-2H3 cells
- Dynamics of granule trafficking
- Cell morphology transitions
- F-actin remodeling
- Cells grown on coverglass
- Cell fixation with 4% (wt/v) paraformaldehyde (PFA) at room temperature (RT) for 30 min
- Cell permeabilization with 0.2% (v/v) Triton-X100 for 15 min
- Cell blocking with 1% bovine serum albumin (BSA) dissolved in PBS
- Incubation with primary antibodies for 2 h at room temperature
- Washing cells 5 times with PBS
- Alexa Fluor-conjugated secondary antibodies diluted 1:1000
- Oregon green 488 or Alexa 546 conjugated phalloidin diluted 1:2000 to stain F-actin
- DAPI (4′, 6-diamidino-2-phenylindol) to stain nuclei
- Cells mounted on glass slides with ProLong™ Gold Antifade mounting media
- Imaging with Zeiss Observer Z1 microscope with a 63X objective (1.4 NA) and processed using Axiovision 4.8 software
- Live-cell imaging using PerkinElmer Ultra-VIEW VoX spinning disk confocal microscope with a 63X objective (1.4 NA), 10 s imaging interval, and Volocity 6.0 software for recording and analysis
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