DOI: 10.22002/8n96z-0z710
title: MEF stably expressing IRE1α-GFP

description: Cryo-CLEM
description: <strong>Tilt Series Date:</strong> 2016-05-06</p>
<strong>Data Taken By:</strong> Stephen Carter</p>
<strong>Species / Specimen:</strong> Mouse Embryonic Fibroblasts</p>
<strong>Strain:</strong> IRE1α-GFP stable expression</p>
<strong>Tilt Series Settings:</strong>  Single Axis, tilt range: (-60.0°, 60.0°), step: 1°, constant angular increment, dosage: 130.0 eV/Å²,  defocus: -7.0 μm,  magnification: 18000x. </p>
<strong>Microscope:</strong> Caltech Polara</p>
<strong>Acquisition Software:</strong> SerialEM</p>
<strong>Upload Method:</strong> reupload_downloaded</p>
<strong>Processing Software Used:</strong> IMOD</p>
<strong>Collaborators and Roles:</strong> Peter Walter, Han Tran</p>
<strong>Purification / Growth Conditions / Treatment:</strong> Cultured in high glucose DMEM media supplemented with 10% tetracycline-free fetal bovine serum (FBS; Takara Bio), 6 mM L-glutamine, and 100 U/ml penicillin/streptomycin.</p>
<strong>Sample Preparation:</strong> Prior to cell plating, gold Quantifoil London finder grids (EMS R2/2 LF-Au-NH2) were UV treated to sterilize and coated with cell adhesion matrix. For MEFs-IRE1-mNG cell line, grids were coated in droplets of 500 ug/mL Fibronectins (Sigma-Aldrich S5171-.5MG) for 5 minutes on each side, washed in PBS, blotted and air-dried. For U2OS-IRE1-mNG cell line, grids were coated with ~4 mg/mL undiluted Collagen type I (Corning 354236) droplets for 20 minutes, washed in PBS, blotted and air-dried. Cells were seeded at 15% confluence and allowed to adhere for 8 hours and induced with Doxycycline (500 nM) for 6 or 18 hours for MEFs and U-2 OS cell lines, respectively. ER stress was then induced by treatment with 1.5 ug/mL of Tunicamycin for 2 hours.</p>
description: Files available via S3 at https://sdsc.osn.xsede.org/ini210004tommorrell/tomography_archive/scd2016-05-06-7/rawdata/</p>tomo6.mrc,  Tilt Series (Pixel Size 6.167 nm), 3.2 GB  
        
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        tomo6.rec,  Reconstruction (Pixel Size 24.668 nm), 890.9 MB  
        
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        tomography6newstack_full.rec,  Reconstruction (Pixel Size 0.0 nm), 577.8 MB  
        
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        tomographysirt6.rec,  Reconstruction (Pixel Size 0.0 nm), 3.3 GB  
        
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        tomographysirt6clip.rec,  Reconstruction (Pixel Size 0.0 nm), 3.3 GB  
        
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