Insect Cell Protocol for LRRK1 and LRRK2 Expression

Pre‐chill centrifuge to 4°C.

Retrieve 1 vials containing 1mL of ~2x10⁷ SF9 cells from liquid N₂ storage.

Thaw vial in hands until ice pellet just disappears, then spray the outside of the tube with 70% EtOH and place in biosafety cabinet, keep everything sterile

Transfer vial of cells (~1mL total) to 15-ml tube, then add 10mL SF900 II media.

Spin at 1000rpm,4°C.

Remove supernatant, add fresh media to 10mLand transfer to 125-ml flask.

Move flask to sticky shaker 90rpm.

Let it grow for 3‐4 days. Count the cells to check concentration.

Once cells have started dividing and reached >2 x 10⁶, add fresh medium and split to ~1.5 x 10⁶ cells/ml.

Transfer cells to 500-mL shaking flask with a volume of at least 100mL when splitting.

Maintain cells so that they are around 1‐2 x 10⁶ cells/ml.

Transform 50µL of DH10EmBacY chemical‐competent cells with 100ng of your miniprepped plasmid DNA. Incubate 27On ice for 0h 30m 0s.

Heat shock at 42°C for 0h 0m 15s.

Chill immediately 42On ice for 0h 2m 0s.

Add 1mLof SOC medium (42Room temperature) and transfer to 14-mL falcon tube.

Shake cells at 220rpm 0h 2m 0s (or at least for 5h 0m 0s).

Plate 12µL , 20µLand 200µL of the transformation on LB‐Kan/Gen/Tet +IPTG +BluoGal Plate.

Wait 2 ‐ 3 days for WHITE colonies to appear. Color change will not happen until day 2 or so.

Screen the white colonies for the presence of all the chains using colony PCR (skip if you are expressing one protein). Grow at least 3 colonies 5h 0m 0s in 6mL LB culture containing 50μg/ml of kanamycin, 7 of gentamicin and 10 of tetracycline and additional antibiotics in plasmid.

Spin down overnight culture in floor centrifuge for 3500rpm.

Resuspend cell pellets in 300µL of buffer P1 from the Qiagen miniprep kit, and transfer to a microfuge tube.

Add 300µL buffer P2 to each tube. Incubate for 0h 5m 0s.

Add 400µL chilled buffer P3 to each tube. Incubate 37On ice for 0h 6m 0s.

Centrifuge for 0h 10m 0sat maximum speed at 4°C.

Transfer supernatant to a new centrifuge tube and centrifuge for 0h 10m 0s at max speed at 4°C.

While centrifuging, add 800µL ice‐cold isopropanol to STERILE 2-mL Eppendorf tube.

After the spin is done, remove supernatant and add into 2-mL Eppendorf tube containing 800µL isopropanol. Incubate for 1h 0m 0s``4On ice.

Spin at maximum speed for 0h 10m 0s at 4°C. Remove supernatant.

Wash pellet with 800µLcold (-20°C) 70% ethanol, invert tube, spin at maximum speed for minutes at 4°C.

Remove ethanol, repeat ethanol wash.

Wash pellet with 800µLcold (-20°C) 70% ethanol. (1/2)

Wash pellet with 800µLcold (-20°C) 70% ethanol. (2/2)

After third wash, remove supernatant and transfer microfuge tube containing precipitated DNA to the hood. Leave cap off of tube and let evaporate for > 0h 20m 0s.

(I usually leave for 0h 30m 0s just to be extra.)

Resuspend the pellet with 30µL to 50µLof nuclease free water and gently flick to mix.

Measure the concentration using nanodrop.

Prepare 4mL of insect cells at 0.5 x10⁶ for each construct. Distribute 2mL each into two 6‐well plate. Leave it at 4Room temperature for at least 0h 10m 0s for cells to adhere.

Allow the vial of Fugene Transfection Reagent to reach 4Room temperature .

Mix by inverting so that there is no precipitate.

To a total volume of 426.8µL, add 4.4µg of bacmid DNA (so 2µgfinal in each well). For DNA concentration of 1,

• 4.4µL of 1DNA
• 422.4µL of insect cell media

To the above DNA in insect cell media, add 13.2µL of Room temperatureFugene Transfection Reagent. Add the transfection reagent directly to the middle of media without touching the side the tube. Mix carefully by tapping at least 10 times. Incubate 0h 15m 0s at Room temperature.

About 0h 10m 0s of incubation Fugene and DNA, remove media from the well. Add fresh of medium to the cells.

Add 200µL of solution from Step 35 dropwise to each well containing 0.8mL cells for a given baculovirus. This will result in 2µg of DNA per well and 6µLof Fugene (hence 3:1 Transfection Reagent:DNA ratio). Swirl the plates gently to mix.

Incubate in 27°Cinsect cell incubator for 24h 0m 0s.

After 24 hours, add additional 1mLof medium and incubate for 2 more days at27°C.

Check the transfection using YFP signal. If cells are more than 30-50% transfected (expressing YFP) , harvest the supernatant within the wells, spin for 1000rpm,4°C, and store the supernatant (v0) at 4°C in the dark.

Add 1‐2% the total volume of previous virus generation to the flask with insect cells at 1x10⁶.

Also, set up control flask to compare.

Incubate in 27°C sticky shaker for 90rpm.

After 3 days, assess quality of baculoviruses by visualizing cells .

Harvest virus only if the cells show YFP signal and swelling. Harvest the supernatant, Spin for 1000rpm,4°C, and store the supernatant (virus) at 4°C in the dark.

To check for protein production and virus stability, save of 1x106 culture (for culture, use less volume accordingly) for gel 100µL of 1x10⁶culture (for culture, use less volume accordingly) for gel, spin it down max speed for 0h 5m 0s, discard supernatant. Resuspend cell pellet in 15µL of 4x sample buffer, 6µL of 10x reducing agent and 39µL water and boil for 0h 10m 0s at 95°C. Load 10µL into the gel.

Divide the cell culture into appropriate conical tubes (15 mL or 50 mL) or JA10 tube (#355605, max volume 465 mL).

Spin at 3500rpm (for conical tubes) or for 0h 17m 0s(for JA10 tubes) at 4°C.

Pour off supernatant into a container with bleach.

Add PBS (~10mL for initial 400mL culture) to one of the conical tubes. Resuspend pellets in PBS and store in conical tube.

Repeat the above step until all cell pellets are resuspended (we usually do 1x 50 mL conical tube for each 400mL growth for ease of protein purification down the road).

Spin at 3500rpm,4°C.

Pour off supernatant.

Label the tube with virus numbers, date harvested and initial cell culture volume.

Flash liquid nitrogen and store the pellets at -80°C.