Cell culture, infection and vitrification

Cell culture and infection experiments as performed in:

Wolff et al. 2020 Science. DOI: 10.1126/science.abd3629

Materials______________________________________________________________________

• EM grids, Quantifoil Au QF R1/4 200 Mesh (Quantifoil Micro Tools, Jena, Germany)
• Glass petri dish
• Whatman™ qualitative filter paper grade 1
• Chloroform
• Glow discharger (K950X evaporator with K350 attachment (EMITECH-Quorum Technologies, Kent, UK)
• 3.5 cm Ø cell culture dishes (Greiner Bio-One, Frickenhausen, Germany)
• Phosphate-buffered saline (PBS)
• EM-grade paraformaldehyde (PFA)
• Cell Culture Flasks (Greiner Bio-One, Frickenhausen, Germany)
• Dulbecco’s modified Eagle’s medium (DMEM; Lonza, Basel, Switzerland)
• Fetal calf serum (FCS; Alkmaar, The Netherlands)
• Tryptose phosphate broth (Life Technologies, Carlsbad, California, USA)
• Diethylaminoethyl (DEAE)-dextran
• Trypsin for cell culture
• 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)
• Heptad repeat 2 (HR2) peptide ((1); GenScript, Piscataway, New Jersey, USA)
• CASY cell counter (Schärfe System GmbH, Reutlingen, Germany)
• Leica EM GP (Leica microsystems, Wetzlar, Germany) plunge freezer
• Heating plate
• Tweezers, style 5/15 (Dumont, Montignez, Switzerland)

Biological materials:

• For Mouse Hepatitis Virus (MHV) experiments:
  • wild type (wt) Mouse Hepatitis Virus (MHV) (ATTC VR-764)
  • 17 clone 1 cells (gift from Stuart Siddell, University of Bristol; maintained in DMEM supplemented with 8% FCS and 8% tryptose phosphate broth)
• For Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) experiments:
  • SARS-CoV-2 isolate BetaCoV/Australia/VIC01/2020 (GeneBank ID: MT007544.1; kindly provided by Leon Caly and Julian Druce, Doherty Institute, Melbourne, Australia (2))
  • Vero E6 cells (ATCC CRL-1586; maintained in DMEM supplemented with 8% FCS)

Protocol______________________________________________________________________

EM grid preparation & cell seeding

• place R1/4 gold Quantifoil grids on a chloroform soaked filter paper in a sealed glass dish and leave overnight
• open glass dish in a fume hood and let chloroform evaporate
• glow-discharge the grids for 40 s at 25 V and 2 × 10^-1 bar
• place the grids in 3.5 cm diameter cell culture containing 1.5 – 2 ml PBS
• sterilize the cell culture dishes for 30 min by UV-sterilization in a laminar-flow cabinet.
• trypsinize, resuspend and count 17 clone 1 or Vero E6 cells cultured in regular cell culture flasks
• prepare a cell suspension with 55,000 cells/ml in the respective DMEM cell culture medium
• remove the PBS of the grids containing 3.5 cm Ø cell culture dishes
• add 2 ml of cell suspension to each dish
• incubate overnight at 37 °C

MHV Infections

• prepare 0.5 ml inoculum per 3.5cm Ø cell culture dish: PBS containing 50 µg/ml DEAE-dextran and 2 % FCS and the respective amount of virus stock to reach an multiplicity of infection (MOI) of 5-10
• wash cells 1x with PBS containing 50 µg/ml DEAE-dextran and 2 % FCS
• add 0.5 ml of inoculum per well
• incubate on a rocking platform in a cell incubator at 37 °C for 1 h
• after 1 h, remove inoculum and add 2 ml of the respective cell culture medium + 25 mM HEPES + 1 µM HR2-peptide

Plunge freezing

• at 8 h post infection (h.p.i.) for MHV
• prepare automatic plunge freezer, set chamber to 37 °C and 95% humidity
• set up a heating plate to 37 °C and place EM grids containing cell culture dishes on them while plunge freezing
• pick up EM grids, transfer to the tweezers of the plunge freezer and add 2-3 µl cell culture medium to avoid drying out
• apply 15 s of blotting from the backside of the grid and 1s post-blotting before automatic plunging
• store the grids at LN₂ temperature until further use

SARS-CoV-2 samples

• Under biosafety level 3 containment
• infect VeroE6 cells with SARS-CoV-2 in the same manner as described above
• chemically fix at 8 h p.i. with 3% PFA in cell culture medium for 1 h
• replace original fixative by 0.5% PFA in cell culture medium
• store overnight at 4 °C
• export samples from the biosafety level 3 facility and let adjust to room temperature
• 30 min prior to plunge-freezing exchange 0.5% PFA containing medium with PFA-free cell culture medium (RT).
• plunge freeze in liquid ethane with the automatic plunge freezer set to 10 s blotting, 2 s post-blotting time and chamber conditions of 21 °C and 90% humidity
• store grids in liquid nitrogen until further use

References______________________________________________________________________

1.         B. J. Bosch, R. van der Zee, C. A. de Haan, P. J. Rottier, The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J. Virol. 77, 8801-8811 (2003).

2.         L. Caly, J. Druce, J. Roberts, K. Bond, T. Tran, R. Kostecki, Y. Yoga, W. Naughton, G. Taiaroa, T. Seemann, M. B. Schultz, B. P. Howden, T. M. Korman, S. R. Lewin, D. A. Williamson, M. G. Catton, Isolation and rapid sharing of the 2019 novel coronavirus (SARS-CoV-2) from the first patient diagnosed with COVID-19 in Australia. Med. J. Aust. 212, 459-462 (2020).