Page 23 - Macromolecular Crystallography

12 MACROMOLECULAR CRYS TALLOGRAPHY

Protocol 1.6 Storing insect cells

Materials 2. Transfer the supernatant to a sterile tube.
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Insect cell culture at 1–2 × 10 cells/ml 3. Keep the cell pellet on ice.
EX-CEL 405™serum-free medium for insect cells containing 4. Resuspend the pellet in 10% dimethyl sulphoxide and
gentamycin sulphate (50 µg/ml), amphotericin B 90% medium (2 volumes conditioned medium +1 volume
fresh medium). Cell density should be about 5 × 10 6
(2.5 µg/ml), and fetal calf serum (10%)
cells/ml.
Sterile cryovials
5. Dispense into cryovials (1 ml/vial).
Liquid nitrogen storage dewar
6. Enclose the cryovials in a vial holder or small polystyrene
A small polystyrene box or a cryovial holder
box.
Dimethyl sulphoxide 7. Transfer the box to –80 C and leave overnight.
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50 ml sterile centrifuge tubes 8. Transfer to liquid nitrogen storage dewar.
Method
1. Harvest cells from a culture containing 1–2 × 10 6
cells/ml by centrifugation at 1000 g for 10 min.

Protocol 1.7 Cotransfection of insect cells to produce recombinant Baculovirus
Materials 7. Pipette 3 ml fresh medium onto plate number 1
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Sf 9 cell culture at 1–2 × 10 cells/ml (control plate).
1 µg linearized BaculoGold™ DNA (BD Biosciences) 8. Add 1 ml of buffer B to the mixture prepared in
Recombinant Baculovirus transfer vector containing the Step 5.
9. Pipette 1 ml of buffer A onto plate number 2.
insert
10. Add the solution from Step 8 drop wise to plate
60-mm tissue culture plates
number 2 and rock the plate gently.
EX-CEL 405™ medium for insect cells containing 10% fetal
11. Incubate both plates at 27 C for 4 h.
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calf serum
12. After 4 h aspirate the medium from plate number 2,
Transfection buffer A and B set (BD Biosciences) wash the cell monolayer with 3 ml of fresh medium by
Sterile microcentrifuge tubes rocking the plate gently, and remove the medium.
Sterile pipettes and pipette tips 13. Add 3 ml of fresh medium to plate number 2 and
SDS-polyacrylamide gel electrophoresis system incubate the plate at 27 C for 4–5 days.
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14. After 4 days compare the two plates for infection.
Infected cells are larger than the uninfected, have
Method enlarged nuclei, stop dividing, and become detached from
1. Prepare two 60-mm tissue culture plates. the surface of the plate. The cells on plate number 1
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2. Pipette 2 × 10 Sf 9 cells onto each plate. should remain uninfected.
3. Place the plates on a level surface. 15. After 5 days transfer the supernatant to sterile
4. Allow the cells to adhere to the bottom of the plate centrifuge tubes. The supernatant of plate number 2
to form a monolayer (40–45 min). contains the recombinant virus.
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5. Mix 0.5 µg linearized BaculoGold™ DNA with 16. Store the recombinant virus at 4 C in a dark place.
2–5 µg recombinant Baculovirus transfer vector 17. Harvest the cells from the plates.
containing the insert and allow the mixture to sit at room 18. Pellet the cells by centrifugation at 2500 g for 5 min.
temperature for 5 min. 19. Analyse the cell pellets from both plates for expression
6. Aspirate the medium from the plates. of the recombinant protein by SDS-PAGE.