TITLE: Insect Cell Protocol for LRRK1 and LRRK2 Expression
AUTHORS: Yu Xuan Lin, Mariusz  Matyszewski

[DESCRIPTION]
Protocol for expressing LRRK1 and LRRK2 in insect cells.

As used in Snead, Matyszewski, Dickey et al. 2022.

[STEPS]
SECTION: Starting Insect Cell Culture from frozen stock
1. 
 
Pre‐chill centrifuge to 4 °C.

SECTION: Starting Insect Cell Culture from frozen stock
2. Retrieve 1 vials containing 1 mL of ~2x107 SF9 cells from liquid N2 storage.

SECTION: Starting Insect Cell Culture from frozen stock
3. 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
SECTION: Starting Insect Cell Culture from frozen stock
4. Transfer vial of cells (~1 mL total) to 15-ml tube, then add 10 mL SF900 II media.

SECTION: Starting Insect Cell Culture from frozen stock
5. Spin at 1000 rpm, 5 min, 4 °C.

SECTION: Starting Insect Cell Culture from frozen stock
6. Remove supernatant, add fresh media to 10 mLand transfer to 125-ml flask.

SECTION: Starting Insect Cell Culture from frozen stock
7. Move flask to sticky shaker  .

SECTION: Starting Insect Cell Culture from frozen stock
8. Let it grow for 3‐4 days. Count the cells to check concentration.
SECTION: Starting Insect Cell Culture from frozen stock
9. Once cells have started dividing and reached >2 x 106, add fresh medium and split to ~1.5 x 106 cells/ml.
SECTION: Starting Insect Cell Culture from frozen stock
10. Transfer cells to 500-mL shaking flask with a volume of at least 100 mL when splitting.

SECTION: Starting Insect Cell Culture from frozen stock
11. Maintain cells so that they are around 1‐2 x 106 cells/ml.
SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
12. Transform 50 µL of DH10EmBacY chemical‐competent cells with 100 ng of your miniprepped plasmid DNA. Incubate on ice for 30 min.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
13. Heat shock at 42 °C for 15 s.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
14. Chill immediately on ice for 2 min.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
15. Add 1 mLof SOC medium (42 Room temperature) and transfer to 14-mL falcon tube.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
16. Shake cells at   2 min (or at least for 300 min).

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
17. Plate 12 µL , 20 µLand 200 µL of the transformation on LB‐Kan/Gen/Tet +IPTG +BluoGal Plate.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
18. Wait 2 ‐ 3 days for WHITE colonies to appear. Color change will not happen until day 2 or so. 
SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
20. Spin down overnight culture in floor centrifuge for 3500 rpm, 5 min.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
21. Resuspend cell pellets in 300 µL of buffer P1 from the Qiagen miniprep kit, and transfer to a microfuge tube.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
22. Add 300 µL buffer P2 to each tube. Incubate for 5 min.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
23. Add 400 µL chilled buffer P3 to each tube. Incubate on ice for 6 min.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
24. Centrifuge for 10 minat maximum speed at 4 °C.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
25. Transfer supernatant to a new centrifuge tube and centrifuge for 10 min at max speed at 4 °C.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
26. While centrifuging, add 800 µL ice‐cold isopropanol to STERILE 2-mL Eppendorf tube. 
 

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
27. After the spin is done, remove supernatant and add into 2-mL Eppendorf tube containing 800 µL isopropanol. Incubate for 60 minon ice. 
SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
29. Wash pellet with 800 µLcold (-20 °C) 70% ethanol,  invert tube,  spin at maximum speed for minutes  at  4 °C.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
30. Remove ethanol, repeat ethanol wash.
SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
31. After third wash, remove supernatant and transfer microfuge tube containing precipitated DNA to the hood. Leave cap off of tube and let evaporate for > 20 min.  
(I usually leave for 30 min just to be extra.)




SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
32. Resuspend the pellet with 30 µL to 50 µLof nuclease free water and gently flick to mix. 
 

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
33. Measure the concentration using nanodrop. 
 

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
19. 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 300 min in 6 mL LB culture containing 50 μg/ml of kanamycin, 7 μg/ml of gentamicin and 10 μg/ml of tetracycline and additional antibiotics in plasmid.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
30.1. Wash pellet with 800 µLcold (-20 °C) 70% ethanol. (1/2)
SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
30.2. Wash pellet with 800 µLcold (-20 °C) 70% ethanol. (2/2)
SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
34. 
 
Prepare 4 mL of insect cells at 0.5 x106 for each construct. Distribute 2 mL each into two 6‐well plate. Leave it at 4 Room temperature for at least 10 min for cells to adhere.

SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
35. Allow the vial of Fugene Transfection Reagent to reach 4 Room temperature .
Mix by inverting so that there is no precipitate. 
SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
36. To a total volume of 426.8 µL, add 4.4 µg of bacmid DNA (so 2 µgfinal in each well). For DNA concentration of 1 µg/µL, 
4.4 µL of   1 µg/µLDNA 
422.4 µL of insect cell media
SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
37. To the above DNA in insect cell media, add 13.2 µL 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 15 min at Room temperature.

SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
38. About 10 min of incubation Fugene and DNA, remove media from the well. Add fresh of medium to the cells. 
 

SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
39. Add 200 µL of solution from Step 35 dropwise to each well containing 0.8 mL cells for a given baculovirus. This will result in 2 µg of DNA per well and 6 µLof Fugene (hence 3:1 Transfection Reagent:DNA ratio). Swirl the plates gently to mix. 
 


SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
40. Incubate in 27 °Cinsect cell incubator for 1440 min.

SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
41. After 24 hours, add additional 1 mLof medium and incubate for 2 more days at27 °C.

SECTION: Transfecting insect cell using Fugene Transfection Reagent (generating V0)
42. Check the transfection using YFP signal. If cells are more than 30-50% transfected (expressing YFP) , harvest the supernatant within the wells, spin for 1000 rpm, 5 min, 4 °C, and store the supernatant (v0) at 4 °C in the dark.

SECTION: Subsequent baculovirus generation (V1 onwards)
43. 
 
Add 1‐2% the total volume of previous virus generation to the flask with insect cells at 1x106. 
SECTION: Subsequent baculovirus generation (V1 onwards)
44. Also, set up control flask to compare.
SECTION: Subsequent baculovirus generation (V1 onwards)
45. Incubate in 27 °C sticky shaker for  .

SECTION: Subsequent baculovirus generation (V1 onwards)
46. After 3 days, assess quality of baculoviruses by visualizing cells .
 

SECTION: Subsequent baculovirus generation (V1 onwards)
47. Harvest virus only if the cells show YFP signal and swelling. Harvest the supernatant, Spin for 1000 rpm, 5 min, 4 °C, and store the supernatant (virus) at 4 °C in the dark. 
 

SECTION: Subsequent baculovirus generation (V1 onwards)
48. To check for protein production and virus stability, save 100 µL of 1x106 culture (for culture, use less volume accordingly) for gel, spin it down max speed for 5 min, discard supernatant. Resuspend cell pellet in 15 µL of 4x sample buffer, 6 µL of 10x reducing agent and 39 µL water and boil for 10 min at 95 °C. Load 10 µL into the gel. 

SECTION: Harvesting Large Insect Cell Culture for Purification
49. 
 
Divide the cell culture into appropriate conical tubes (15 mL or 50 mL) or JA10 tube (#355605, max volume 465 mL).
SECTION: Harvesting Large Insect Cell Culture for Purification
50. Spin at 3500 rpm, 5 min (for conical tubes) or for 17 min(for JA10 tubes) at 4 °C.

SECTION: Harvesting Large Insect Cell Culture for Purification
51. Pour off supernatant into a container with bleach.
SECTION: Harvesting Large Insect Cell Culture for Purification
52. Add PBS (~10 mL for initial 400 mL culture) to one of the conical tubes. Resuspend pellets in PBS and store in conical tube.

SECTION: Harvesting Large Insect Cell Culture for Purification
53. Spin at 3500 rpm, 10 min, 4 °C.
SECTION: Harvesting Large Insect Cell Culture for Purification
54. Pour off supernatant.
SECTION: Harvesting Large Insect Cell Culture for Purification
55. Label the tube with virus numbers, date harvested and initial cell culture volume.
SECTION: Harvesting Large Insect Cell Culture for Purification
56. Flash liquid nitrogen and store the pellets at -80 °C.

SECTION: Preparing genomic DNA using chemical transformation to transfect insect cell
28. Spin at maximum speed for 10 min at 4 °C. Remove supernatant.

SECTION: Harvesting Large Insect Cell Culture for Purification
52.1. Repeat the above step until all cell pellets are resuspended (we usually do 1x 50 mL conical tube for each 400 mL growth for ease of protein purification down the road).