Contents

• Plastic consumables required for baculovirus work
• Medium
• Baculovirus Reagents
• Reviving Sf9 cells stored in liquid nitrogen
• Routine subculture
• Freezing Sf9 cells for storage in liquid nitrogen
• Counting Sf9 cells
• Transfection of Sf9 cells with recombinant bacmid DNA
• Harvest/storage of recombinant baculovirus
• Amplification
• Plaque assays
• Infecting Sf9 cells for production of recombinant protein
• Analyzing recombinant protein expression by cells grown in shake flasks
• Preparation of extract

Reviving Sf9 cells stored in liquid nitrogen

1. Rapidly thaw frozen cryovial of cells in 28°C waterbath with shaking.
2. Spray cryovial with 70% ethanol, wipe dry and transfer to hood.
3. Cut cryoflex with scalpel, and unscrew top.
4. Use a sterile pasteur pipette to transfer contents of vial to 9 ml "ready to use" medium.
5. Spin down cells at 1,000x g for 5 mins.
6. Discard supernatant.
7. Resuspend cell pellet in 20 mls "ready to use" medium and transfer to 125 ml flask.
8. Incubate cells at 28°C with shaking at 150 rpm for 48 hours.
9. After 48 hours, count cells and subculture.

Routine subculture

1. Grow cells to mid log 1 - 2 x 10⁶ cells/ml.
2. Split cells, by diluting with "ready to use" medium to 2 x 10⁵ cells/ml (ensure lids are loose!).
3. Sf9 cells can be grown routinely in medium occupying up to 1/4 of the vessel volume, e.g. 500ml per 2L roller bottle.
4. Continue incubation at 28°C with shaking at 150 rpm.
5. Spin down cells once a fortnight and resuspend in fresh "ready to use" medium.

Routine growth of Sf9 cells in suspension culture

Freezing Sf9 cells for storage in liquid nitrogen

1. Prepare cryopreservation medium as follows: 7.5% DMSO, 46.25% "ready to use" SFM, 46.25% conditioned SFM (medium used for growing Sf9 cells for 2-3 days, sterile filtered) as appropriate and store on ice.
2. Harvest cells in mid log growth phase by centrifuging at 1000x g for 5 mins.
3. Discard supernatant and store pellet on ice.
4. Resuspend cells in cold cryopreservation medium to a final density of 2 x 10⁷ cells/ml.
5. Aliquot 1ml volumes of cells into 1 ml internal thread cryovials.
6. Seal cryovials with cryoflex as follows:
   1. Place cryovial in cryoflex, with the cryoflex cut to allow a 1 cm overlap at each end of the cryovial.
   2. Briefly rotate the cryovial in a Gaz flame to shrink the cryovial.
   3. Use fine forceps to pinch together and seal the cryoflex as close to the ends of the cryovial as possible.
   4. Place cryovial on ice.
   5. When all cryovials are sealed, use a pair of scissors to trim the cryoflex as close to the top and bottom of the cryovial, without breaking the seal.
   6. Place cryovials in Scotlab polystyrene box at -20°C for 1 hour.
   7. Place box at -80°C overnight.
   8. Place cryovials in liquid nitrogen store.

Counting Sf9 cells using an improved Neubauer haemocytometer

1. Combine and mix the following: 0.5ml 0.4% trypan blue + 0.3 ml 1x PBS + 0.2 ml Sf9 cells.
2. Dampen the haemocytometer, either side of the trough, and press coverslip firmly into place. Check for Newton's rings.
3. Mix the cell suspension again and then carefully transfer a few µls by placing the end of a filled micropipette tip onto the surface of the haemocytometer adjacent to cover slip. The cell suspension will flow under the cover slip by surface tension.
4. Repeat for second haemocytometer chamber.
5. Number of cells/ml = Average number of cells / Large Square x 10⁴ x 5

Transfection of Sf9 cells with recombinant bacmid DNA

1. Seed 9 x 10⁵ cells per 35 mm well (of a 6 well plate) in 2 ml of SF900II SFM containing penicillin/streptomycin at 0.5 x final concentration (30µg/ml sodium benzylpenicillin, 50µg/ml streptomycin sulphate).
2. Allow cells to attach at 28°C for at least 1 hour.
3. Prepare the following solutions.
   1. For each transfection, dilute 5 µl of mini-prep DNA into 100 µl SF900II SFM without antibiotics.
   2. For each transfection, dilute 6 µl Cellfectin reagent into 100 µl SF900II without antibiotics. Note: Cellfectin reagent is a lipid suspension that may settle with time.
4. Mix thoroughly by inverting the tube 5 - 10 times before removing a sample for transfection to ensure that a homogeneous sample is taken.
5. Combine the two solutions, mix gently, and incubate for 15 to 45 mins at room temperature.
6. Wash the cells once with 2 ml of SF900II SFM without antibiotics.
7. For each transfection, add 0.8 ml of SF900II SFM to each tube containing the lipid-DNA complexes. Mix gently. Aspirate wash media from cells and overlay the diluted lipid-DNA complexes onto the cells.
8. Incubate cells for 5 hours in a 28°C incubator.
9. Remove the tranfection mixtures and add 2 ml of SF900II SFM containing antibiotics. Incubate cells in a 28°C incubator for 72 hours.
10. Infected and uninfected Sf9 cells can be distinguished by morphology (see figures below).

    Uninfected Sf9 cells. These cells continue to divide and form a confluent monolayer.	Sf9 cells infected with recombinant baculovirus. These cells stop dividing and enlarge.

11. Harvest virus from cell culture medium at 72 hours post-transfection.

Harvest/storage of recombinant baculovirus

1. When harvesting virus from the transfection, transfer the supernatant (2 ml) to a sterile, capped tube. Clarify by centrifugation for 5 min at 1000 x g and transfer the virus-containing supernatant to a fresh tube.
2. From the initial tranfection, viral titers of 2 x 10⁷ to 4 x 10⁷ pfu/ml can be expected.
3. Store the virus at 4°C, protected from light. For long term storage of virus, the addition of fetal bovine serum (FBS) to a final concentration of at least 2% FBS is recommended. Storage of an aliquot of the viral stock at -70°C is also recommended. Stability studies have shown that, with two specific exceptions, the viability declines very little over the period of a year's storage at 4°C (see first figure below). Two specific viruses (BRAF35 and hXTH2) were very unstable at 4°C, with over 1000 fold loss of titer in 3 months(see second figure below). These viruses appeared stable when stored at -70°C. A single freeze/thaw cycle does not decrease viability (see third figure below).
4. Determine the viral titer before amplifying the virus stock or analyzing protein expression.

Amplification

Only use Sf9 cells grown in SF900II for amplification. Infect a suspension or monolayer culture at a Multiplicity of Infection (M.O.I.) of 0.01 to 0.1. Use the following formula: Inoculum required (ml) = desired MOI x total number of cells / titer of viral inoculum (pfu/ml)

Transfections generally yield > 1 x 10⁷ pfu/ml, allowing use of just part of the P1 stock to be amplified with suspension cells at 2 x 10⁶ cells/ml. The Quick Amplification Guide suggests the best way to amplify, should the baculovirus yield be lower than average.

Quick Amplification Guide

1. Efficient viral amplification requires extra aeration. Do not exceed 1/20th vessel volume and use Erlenmeyer flasks rather than roller bottles.
2. Continue incubation at 28°C, 150 rpm for 72 hours. The titer reaches a maximum at 72 hours and then declines (see figure below).

Plaque assays

The plaque assay can be used to plaque purify virus or to determine viral titer in plaque-forming units per ml (pfu/ml) so that known amounts of virus can be used to infect cells during subsequent experimental work. In this assay, cell monolayers are infected with a low ratio of virus, such that only isolated cells become infected. An overlay of agarose keeps the cells stable and limits the spread of virus. When each infected cell produces virus and eventually lyses, only the immediate neighboring cells become infected. Areas of clearing are produced, called plaques. Each plaque represents a single virus. Therefore, clonal virus populations may be purified by isolating individual plaques.

Day 1

1. Add agarose (A-4018; Sigma Type VII: low gellingtemperature) to water to give a final suspension equivalent to 3%, and sterilize by autoclaving.
2. Melt 3% agarose in microwave, and place in water bath at 45°C.
3. Count Sf9 cells, and dilute the culture to 5 x 10⁵ cells/ml in "ready to use" medium.
4. Seed 35 mm petri dishes with 2 ml of culture (1 x 10⁶ cells).
5. Incubate the dishes for 2 hours at room temperature.
6. Set up virus dilutions using 20 µl virus in 180 µl "ready to use" medium; use dilutions down to 10^-7.
7. Remove medium from dishes and replace with 100 µl virus dilution.
8. Incubate at room temperature outside the hood for 1 hour. Agitate gently every 15 mins.
9. Remove viral inoculum from 6 dishes.
10. Combine 8 mls "ready to use" medium with 4 mls 3% agarose (and 120 µl X-gal 25 mg/ml in DMF, if colour selection required).
11. Pipette 2 mls of 1% agarose in medium onto each dish.
12. When the agarose is set, overlay with 1ml "ready to use" medium.
13. Disinfect sandwich box with 70% ethanol, and line with tissue dampened with sterile water.
14. Incubate in the humidified sandwich box at 28°C for 3 - 4 days.

Day 4

1. Dilute 1 part 0.4% neutral red with 19 parts 1x PBS.
2. Stain plaques by adding 1 ml neutral red/PBS solution to each dish. Incubate at 28°C in the dark for 2 hours.
3. Aspirate liquid and invert dishes overnight (leave at room temperature in the dark).

Day 5

1. Count plaques!

Baculovirus plaques. Zones of clearing (plaques) are generated by infection of Sf9 cells with individual baculovirus particles. Uninfected Sf9 cells surrounding the plaque are stained pink with neutral red.

Infecting Sf9 cells for production of recombinant protein

1. Grow Sf9 cells to a density of approximately 2 x 10⁶ cells/ml (500ml culture/2l roller bottle, for large scale expression).
2. Infect cells with an M.O.I of 2.
3. Incubate infected cells at 28°C with shaking at 150rpm for 1-4 days (generally 3 days).
4. Take samples for analyzing recombinant protein expression (see next section).
5. Harvest cells by centrifugation for 5 mins at 1000x g.
6. Discard supernatant and store pellet at -80°C.
   

Analyzing recombinant protein expression by cells grown in shake flasks

"Whole cell"

Take 3.3 x 10⁵ pelleted Sf9 cells and lyse with 50 µl 1x SDS-GLB. Boil samples for 3 mins and load 10 µl onto an SDS polyacrylamide gel.

Soluble vs insoluble

Take 3.3 x 10⁶ pelleted cells and resuspend in 0.5 ml lysis buffer. Spin. Remove supernatant and mix with an equal volume of 2 x SDS GLB (=soluble fraction). Add 1 ml of 1 x SDS GLB to pellet (=insoluble fraction). Boil samples for 3 mins and load 20 µl onto an SDS polyacrylamide gel.

Preparation of extract

1. Add purification compatible buffer to pellet, using 5 ml buffer per gram of cells.
2. Homogenize (10 strokes).
3. Remove cell debris by centrifugation at 10,000 x g for 10 mins.
4. Transfer the supernatant to a new tube and proceed with purification.