1The Babraham Institute, Laboratory of Molecular Signalling, Cambridge, UK 2Department of Pharmacology, University of Cambridge, Cambridge, UK
Sponsored,vendor-submitted protocol Sponsored by Olympus America Inc. Published in November 2009 (p.43) DOI: 10.2144/000113032 Abstract
By employing an advanced imaging system, total internal reflection fluorescence (TIRF) illumination was used to observe the intracellular distribution of STIM-1 labeled structures at the plasma membrane, near-simultaneously with intracellular calcium concentration.
By employing an advanced imaging system, total internal reflection fluorescence (TIRF) illumination was used to observe the intracellular distribution of STIM-1 labeled structures at the plasma membrane, near-simultaneously with intracellular calcium concentration.Materials
HEK-293 Cells (passage 35-50 from Microbix corporation)
YFP-/mCherry–tagged Constructs (gifts from R.S. Lewis, Stanford University, Palo Alto, CA, USA, and S. Muallem, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USA)
Fura-2 AM (Invitrogen, Carlsbad, CA, USA) diluted to 2 mM stock in DMSO/20% Pluronic (Invitrogen)
Thapsigargin (Sigma, St. Louis, MO, USA) diluted to 2 mM stock in DMSOMethods Cell Preparation
Seed HEK-293 cells onto poly-L-lysine–coated 16 mm diameter round glass coverslips and transfect with YFP-/mCherry–tagged constructs (1 µg DNA per well) using jetPEI (Polyplus transfection) 16 h prior to imaging.
For calcium imaging, load cells with Fura-2 AM (2 µM) diluted in calcium-containing extracellular (imaging) buffer (121 mM NaCl, 6 mM NaHCO3, 5.4 mM KCl, 5.5 mM D-glucose, 0.8 mM MgCl2, 25 mM HEPES, 1.8 mM CaCl2, pH 7.4) for 30 min.
Remove dye-containing buffer from cells, wash with imaging buffer and leave for a period of 30 min at room temperature to allow for de-esterification.
Mount coverslip in an appropriate imaging chamber (we use custom made stainless steel rings fitted with a teflon retainer) and immerse cells in imaging buffer.
Olympus cell∧R imaging system comprising IX81 microscope, 100×1.45 NA apochromat objective, MT-20 illumination unit, 488 nm/20 mW and 561 nm/25 mW lasers, and Hamamatsu ORCA ER camera.
Solent Scientific environment chamber to maintain temperature at 32°C.
Exciter filters for wide-field epifluorescence images of YFP and Fura-2: 470/40 nm and 380/15 nm, respectively. 495 DCLP and 505 nm long pass emission filter used for both dyes.
YFP TIRF and mCherry TIRF images generated using 488 nm and 561 nm laser excitation, respectively, a multiband dichroic mirror with edge wavelengths at 495 nm and 573 nm and a 527/21 nm, 645/24 nm dual bandpass emission filter.
Exposure times: YFP wide-field, 500 ms; YFP TIRF, 300 ms; mCherry TIRF, 400 ms; Fura-2 AM, 100 ms.
Camera gain set to 100 for all experiments.
Place imaging chamber on microscope stage.
Place vacuum aspirator in imaging chamber ∼5 mm from coverslip surface.
Add 2 ml fresh imaging buffer (pre-heated to 32°C) to cells.
Focus on cells and capture reference images.
After 60 s, apply 4 ml calcium-free buffer.
After a further 60 s, apply 2 µM thapsigargin diluted in calcium-free buffer and continue recording for 10 min.
All image analysis can be done using Olympus cell∧R software.Results
Figure 1 compares both wide-field epifluorescence and TIRF images from STIM1-YFP-transfected HEK cells. Thapsigargin (2 µM) was added at 0 min; bar, 10µm. Acquisition of TIRF images and wide-field epifluorescence images (Figure 2) allows STIM1 translocation to be monitored concomitant with changes in intracellular calcium concentration. Figure 2A shows the sequential acquisition of STIM1-YFP (TIRF), STIM1-mCherry (TIRF) and Fura2 (wide-field epi-fluorescence) images from the same cells. A set of three images (2× TIRF, 1× wide-field) was acquired every 2 s (images shown acquired 5 min after application of thapsigargin). Figure 2B illustrates the change in intracellular calcium concentration of cells outlined in A; bar, 10 µm.
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