Page 22 - Macromolecular Crystallography

CLASSICAL CLONING, EXPRESSION, AND PURIFICATION 11

Protocol 1.5 Starting an insect cell culture

Materials 10. Resuspend the cells in 10 ml fresh medium.
Sf 9 insect cells (BD Biosciences or Invitrogen) 11. Mix 10 µl cell suspension with 10 µl of Trypan Blue
EX-CEL 405™serum-free medium for insect cells (RJH solution and estimate the viable cell density using a
Biosciences) or Sf 900 ll SFM (Invitrogen-GIBCO) haemocytometer. Non-viable cells turn blue.
12. Adjust cell density to 250,000 viable cells/ml medium.
Fetal calf serum (LabClinics SA, Barcelona)
13. Transfer the cell suspension to a 50 ml tissue culture
Gentamycin sulphate, 10 mg/ml stock (BD Biosciences)
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flask and incubate at 27 C or room temperature for 48 h.
Amphotericin B, 250 µg/ml stock (BD Biosciences)
14. After 48 h, examine the flask using a light microscope
Haemocytometer
and reincubate until the cells become confluent.
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27 C incubator 15. Dislodge the cells by tapping the flask gently on a
Tissue culture flasks bench and transfer the cell suspension to a 25 ml universal
Sterile 10 ml pipettes tube.
Sterile Spinner flasks (200 ml and 1 litre), spinner apparatus 16. Pellet the cells by centrifugation at 1000 g for 2–5 min.
Sterile 25 ml and 50 ml plastic tubes 17. Transfer the supernatant to a 50 ml sterile tube and
Sterile plastic Pasteur pipettes add two volumes of fresh medium.
Trypan Blue Stain (0.2%) solution in PBS 18. Resuspend the cell pellet in 10 ml of the medium in
Step 17 and determine cell density.
19. Seed the cells into a new tissue culture or spinner flask
Method
1. Equilibrate the medium at room temperature. at a density of 250,000 cells/ml using the medium in Step
2. Add gentamycin sulphate (50 µg/ml), amphotericin B 17 (some fresh medium may be added if required).
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(2.5 µg/ml), and fetal calf serum (10%). 20. Incubate the flask at 27 C or room temperature for
3. Remove a batch of frozen cells from liquid nitrogen 48 h.
storage dewar. 21. Split or scale up the culture when the cell density
6
4. Thaw the cells quickly by dipping the vial in a water reaches 2 × 10 cells/ml.
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bath at 37 C for about 30 sec. 22. Steps 19–21 are repeated until a required culture
5. Spray the outside of the vial with 70% ethanol and volume is obtained.
place it in a sterile hood.
6. Transfer the cells to a 25 ml sterile universal vial using Insect cells can be adapted to a serum-free insect cell
a sterile plastic Pasteur pipette. medium by slowly decreasing the concentration of fetal calf
7. Add 20 ml medium drop wise. serum in the medium.
8. Centrifuge at 600 g, at room temperature, for 2–5 min. Note: Protocols 1.5 to 1.11 have been adapted from
9. Aspirate the supernatant using a 25 ml sterile pipette Baculovirus Expression System Manual, 6th edn, May 1999
taking care not to disturb the cell pellet. (www. bdbiosciences.com).

culture are transferred for long-term storage in liq- (Protocol 1.7) and normally generate high-titre
uid nitrogen. Properly stored cells (Protocol 1.6) stocks. The viral titre is determined by plaque assay
remain viable for several years. (Protocol 1.8) so that known amounts of the recom-
The plasmid DNA used for cotransfection should binant virus are used in subsequent virus amplifica-
be as pure as possible. Insect cells are sensitive to tion experiments (Protocol 1.9) to produce large viral
impurities in plasmid samples and may lyse before stocks.
the recombinant virus is regenerated, resulting in A small-scale titration experiment is carried out
very low viral titres. For good cotransfection exper- to determine the optimum amount of the recom-
iments, monolayers of healthy cells with an initial binant viral stock required for protein production
confluency of 60–70% are required. using a 6-well tissue culture plate with a monolayer
5
ProceduresforobtainingrecombinantBaculovirus of 6 × 10 cells per well. The wells are infected
using linearized BaculoGold™ DNA are simple with 0, 10, 20, 40, 60, and 80 µl of the recombinant