May 23, 2022
Insect Cell Protocol for LRRK1 and LRRK2 Expression
- Yu Xuan Lin1,
- Mariusz Matyszewski1
- 1Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
Protocol Citation: Yu Xuan Lin, Mariusz Matyszewski 2022. Insect Cell Protocol for LRRK1 and LRRK2 Expression. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzyyrrlx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 11, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 58050
Keywords: insect cells, Sf9, LRRK1, LRRK2, ASAPCRN
Funders Acknowledgement:
ASAP
Grant ID: ASAP-000519
MJFF
Grant ID: 18321
Abstract
Protocol for expressing LRRK1 and LRRK2 in insect cells.
As used in Snead, Matyszewski, Dickey et al. 2022.
Materials
Materials
Starting Insect Cell Culture from frozen stock:
- Sf900 cell stock
- SF900-II Serum media
Preparing genomic DNA using chemical transformation to transfect insect cell:
- DH10EmBacY Cells
- Qiagen Miniprep Kit
- Isopropanol; Ethanol
Transfecting insect cell using Fugene Transfection Reagent (generating V0):
- Fugene Transfection Reagent
- insect cell media
Starting Insect Cell Culture from frozen stock
Starting Insect Cell Culture from frozen stock
5m
5m
Note
- Frozen stocks of sf900 cells contain DMSO, which needs to be removed and replaced with SF900‐II Serum media. The Cells are started at high‐density (~2 x 106 cells/ml) until they start to divide. Subsequently, cultures are maintained at 2‐4 x 106 cells/ml to keep them in log phase growth. Only split cells that are between 2‐5 x 106 cells/ml to 1 x 106 cells/ml.
- For growth over the weekend, dilution to 0.7 x 106 cells/ml on Friday pm and splitting Monday am can work well. Ideally, cells should double every 24‐30 hour. Fresh cultures should be started about every 2 months.
Pre‐chill centrifuge to 4 °C .
Retrieve 1 vials containing 1 mL 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 (~1 mL total) to 15-ml tube, then add 10 mL SF900 II media.
Spin at 1000 rpm, 4°C, 00:05:00 .
5m
Remove supernatant, add fresh media to 10 mL and transfer to 125-ml flask.
Move flask to sticky shaker 90 rpm, 27°C .
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 100 mL when splitting.
Maintain cells so that they are around 1‐2 x 106 cells/ml.
Preparing genomic DNA using chemical transformation to transfect insect cell
Preparing genomic DNA using chemical transformation to transfect insect cell
13h 10m 15s
13h 10m 15s
Transform 50 µL of DH10EmBacY chemical‐competent cells with 100 ng of your miniprepped plasmid DNA. Incubate On ice for 00:30:00 .
30m
Heat shock at 42 °C for 00:00:15 .
15s
Chill immediately On ice for 00:02:00 .
2m
Add 1 mL of SOC medium (Room temperature ) and transfer to 14-mL falcon tube.
Shake cells at 220 rpm, 37°C Overnight (or at least for 05:00:00 ).
5h 2m
Plate 12 µL , 20 µL and 200 µL 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 Overnight in 6 mL LB culture containing 50 μg/ml of kanamycin, 7 undetermined of gentamicin and 10 undetermined of tetracycline and additional antibiotics in plasmid.
5h
Spin down overnight culture in floor centrifuge for 3500 rpm, 00:05:00 .
5m
Resuspend cell pellets in 300 µL of buffer P1 from the Qiagen miniprep kit, and transfer to a microfuge tube.
Add 300 µL buffer P2 to each tube. Incubate for 00:05:00 .
5m
Add 400 µL chilled buffer P3 to each tube. Incubate On ice for 00:06:00 .
6m
Centrifuge for 00:10:00 at maximum speed at 4 °C .
10m
Transfer supernatant to a new centrifuge tube and centrifuge for 00:10:00 at max speed at 4 °C .
10m
While centrifuging, add 800 µL ice‐cold isopropanol to STERILE 2-mL Eppendorf tube.
Note
NOTE: This tube must be sterile since the contents of the tube will be added to insect cells.
After the spin is done, remove supernatant and add into 2-mL Eppendorf tube containing 800 µL isopropanol. Incubate for 01:00:00 On ice .
1h
Spin at maximum speed for 00:10:00 at 4 °C . Remove supernatant.
10m
Wash pellet with 800 µL cold (-20 °C ) 70% ethanol, invert tube, spin at maximum speed for minutes at 4 °C .
Remove ethanol, repeat ethanol wash.
Wash pellet with 800 µL cold (-20 °C ) 70% ethanol. (1/2)
Wash pellet with 800 µL cold (-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 > 00:20:00 .
(I usually leave for 00:30:00 just to be extra.)
50m
Resuspend the pellet with 30 µL to 50 µL of nuclease free water and gently flick to mix.
Note
Note: Do this in the tissue culture hood.
Measure the concentration using nanodrop.
Note
If you can't proceed to transfection, store the DNA in 4 °C for up to 2 weeks.
Transfecting insect cell using Fugene Transfection Reagent (generating V0)
Transfecting insect cell using Fugene Transfection Reagent (generating V0)
10m
10m
Note
Note:
- Everything involving insect cells should be done in the tissue culture hood to avoid contamination.
- There is no antibiotics in the media.
- keep everything as sterile as possible.
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 Room temperature for at least 00:10:00 for cells to adhere.
10m
Allow the vial of Fugene Transfection Reagent to reach Room temperature .
Mix by inverting so that there is no precipitate.
To a total volume of 426.8 µL , add 4.4 µg of bacmid DNA (so 2 µg final in each well). For DNA concentration of 1 undetermined ,
- 4.4 µL of 1 undetermined DNA
- 422.4 µL of insect cell media
To the above DNA in insect cell media, add 13.2 µL of Room temperature Fugene 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 00:15:00 at Room temperature .
15m
About 00:10:00 of incubation Fugene and DNA, remove media from the well. Add fresh of medium to the cells.
Note
Be careful as to not disturb the cells at the bottom of the wells.
10m
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 µL of Fugene (hence 3:1 Transfection Reagent:DNA ratio). Swirl the plates gently to mix.
Note
Note: This step needs optimization. Vary the amount of DNA and the ratio of transfection reagent:DNA.
Incubate in 27 °C insect cell incubator for 24:00:00 .
1d
After 24 hours, add additional 1 mL of 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 1000 rpm, 4°C, 00:05:00 , and store the supernatant (v0) at 4 °C in the dark.
5m
Subsequent baculovirus generation (V1 onwards)
Subsequent baculovirus generation (V1 onwards)
20m
20m
Note
For subsequent virus generation beyond V0, previous virus generation is added at 1‐2% of the total volume of the culture.
V1 = 50 mL total: use 1 mL of V0
V2 = 400 mL total: use 4 mL of V1
(Can be used to harvest cells for protein purification from this point)
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 90 rpm, 72:00:00 .
After 3 days, assess quality of baculoviruses by visualizing cells .
Note
- The transfected cells should swell ~20-100% larger than the control.
- Express YFP signal; at this point, 100% of the cells should express YFP.
- Cells that are transfected should be less concentrated than control after 3 days.
Harvest virus only if the cells show YFP signal and swelling. Harvest the supernatant, Spin for 1000 rpm, 4°C, 00:05:00 , and store the supernatant (virus) at 4 °C in the dark.
Note
Virus can be stored for several months. (about half a year for v0, and a year for v1)
5m
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 00:05:00 , 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 00:10:00 at 95 °C . Load 10 µL into the gel.
15m
Harvesting Large Insect Cell Culture for Purification
Harvesting Large Insect Cell Culture for Purification
32m
32m
Note
This is the V2 stage as described above in 400-mL tubes; For LRRK2 purification, grow 3x 400 mL growths or 2x 600 mL growths. Harvest after 3 days.
Divide the cell culture into appropriate conical tubes (15 mL or 50 mL) or JA10 tube (#355605, max volume 465 mL).
Spin at 3500 rpm, 00:05:00 (for conical tubes) or for 00:17:00 (for JA10 tubes) at 4 °C .
22m
Pour off supernatant into a container with bleach.
Add PBS (~10 mL for initial 400 mL 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 400 mL growth for ease of protein purification down the road).
Spin at 3500 rpm, 4°C, 00:10:00 .
10m
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 .