MEF stably expressing IRE1α-GFP

Description

Technical info

Tilt Series Date: 2016-05-19

Data Taken By: Stephen Carter

Species / Specimen: Mouse Embryonic Fibroblasts

Strain: IRE1α-GFP stable expression

Tilt Series Settings: Single Axis, tilt range: (-66.0°, 66.0°), step: 1°, constant angular increment, dosage: 180.0 eV/Ų, defocus: -5.0 μm, magnification: 41000x.

Microscope: Caltech Polara

Acquisition Software: SerialEM

Upload Method: pipeline

Processing Software Used: IMOD

Collaborators and Roles: Peter Walter, Han Tran

Purification / Growth Conditions / Treatment: 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.

Sample Preparation: 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.

Attached Files

manualtomo1.mrc, Tilt Series (Pixel Size 2.6 nm), 3.8 GB