TITLE: Visualization of LRRK2 filaments in 293T cells
AUTHORS: Eva Karasmanis, Kathryn S Hatch

[DESCRIPTION]
Visualization of LRRK2 filaments in 293T cells
 
GOAL: Express GFP-LRRK2 with or without DARPin E11 and quantify the percentage of cells with LRRK2 filaments in the presence and absence of MLi-2 in 293T cells.

[STEPS]
SECTION: Day 1: fibronectin coating and cell plating
1. Visualization of LRRK2 filaments in 293T cells
 
GOAL: Express GFP-LRRK2 with or without DARPin E11 and quantify the percentage of cells with LRRK2 filaments in the presence and absence of MLi-2 in 293T cells.

Constructs needed: 

1) CMV-8xHISDaprin C12-FLAG
2) CMV- His-daprin E11-FLAG 
3) CMV-GFP-LRRK2 
 

SECTION: Day 1: fibronectin coating and cell plating
2. Fibronectin Coating  (Sigma cat# F0895, 0.1% solution, 1 mg/ml):
SECTION: Day 1: fibronectin coating and cell plating
2.1. Make 0.01 µg/µL solution of fibronectin, stock @ 1 mg/mL For 6x 35 mm dishes (12 ml fibronectin working stock)- 0.12 mL fibronectin + 11.88 mL 1X PBS

SECTION: Day 1: fibronectin coating and cell plating
2.3. Add 2 ml of 10 ug/mL fibronectin per 35 mm dish or 6 well.
SECTION: Day 1: fibronectin coating and cell plating
2.4. Incubate at 37 °C 5% CO2 for 45 min

SECTION: Day 1: fibronectin coating and cell plating
2.5. Wash with PBS and let dry for 45 min in the tissue culture hood (no UV) 

3.1. Dissociate cells, count and plate 6 well plate with 200K cells /well. For transfection, plate in antibiotic-free media (DMEM+10% FBS)

SECTION: Day 2   Transfect cells with GFP-LRRK2 and Darpins:
4. Transfect 800 ng LRRK2 and 400ng Darpin with 5 μL PEI /well. 
- cells should be 50-60% confluent
 In a sterile tube dilute 800 ng GFP-LRRK2 and 400 ng DARPin E11 plasmid DNA in Optimem (150 µL/well) 

Per Well: 
a) 150 µL Optimem (prewarmed)+5 µLPEI 
b) 150 µL Optimem (prewarmed) +800 ng GFP LRRK2 and 400 ng DARPin E11 DNA.
or 
c) 150 µL Optimem (prewarmed) + 800 ngGFP LRRK2 

Add PEI to diluted DNA - (Per reaction: 5 µL of 1 µg/µL stock). Mix immediately.  

SECTION: Day 2   Transfect cells with GFP-LRRK2 and Darpins:
4.2. Incubate at Room temperature for 15 min

SECTION: Day 2   Transfect cells with GFP-LRRK2 and Darpins:
4.3. Add DNA/PEI mixture to cells dropwise.
SECTION: Day 2   Transfect cells with GFP-LRRK2 and Darpins:
4.4. Swirl the plate to distribute Incubate at 37 °C  5% CO2 for 2880 min

13. 2X Rinse with Wash 2X (5 min, Room temperature)  with blocking buffer

14. Add primary antibody mix : 
Add 40uL on parafilm and flip coverslip on the antibody. Incubate 180 min Room temperature or 4 °C 5 min . If incubating overnight, make a humidity chamber before placing in fridge.


HERE: we used 1:200 rabbit polyclonal anti-FLAG DYKDDDDK tag – (ptg labs Cat no : 20543-1-AP) 
15. Rinse 2X with blocking buffer
16. Wash 2X 5 min Room temperature  with blocking buffer

17. Add secondary mix in PBS (1:200 Goat a-rabbit Alexa568; SIGMA A-11011 +1:5000 DAPI**)  45 min Room temperature 
** DAPI can alternatively be added as a separate step at 1:1000 dilution for 15 min
18. 5X Rinse with PBS 1X 
19. Mount in Fluorsave hard media (Millipore 345789)
20. Let dry for at least an hour. Store in Fridge 4oC if not imaging immediately. Check coverslip is set with tweezers before imaging.
SECTION: Imaging and analysis
21. Blind your mounted slides before imaging to prevent bias during aquisition. 
SECTION: Day 1: fibronectin coating and cell plating
3. Plate cells onto Fibronectin coated dishes
SECTION: Day 4 and 5  MLi-2 treatment and Fix/Stain cells
None. Aspirate media
SECTION: Day 4 and 5  MLi-2 treatment and Fix/Stain cells
None.  Fix/Stain  for FLAG : Prewarm 15 mL of 3 % (v/v) PFA, 4 Mass / % volumesucrose in 1X in PBS1X (make fresh each time). -  - PFA stock 16 % (v/v); EMS, Catalog No.50-980-487
- Calculation for sucrose: 4% w/v aka 4 g of sucrose for 100 mL solution. X mL of solution will require Y grams of sucrose => Y g of sucrose = 4g*XmL/100mL  
SECTION: Day 2   Transfect cells with GFP-LRRK2 and Darpins:
4.1. In a sterile tube dilute 800 ng GFP-LRRK2 and 400 ng DARPin E11 plasmid DNA in Optimem (150uL/well) 

Per Well: 
a) 150 µLOptimem (prewarmed)+ 5 µLPEI 
b) 150 µL Optimem (prewarmed) + 800 ng GFP LRRK2 and 400 ng DARPin E11 DNA.
or 
c)150 µL Optimem (prewarmed) + 800 ng GFP LRRK2 

Add PEI to diluted DNA - (Per reaction: 5 µL of 1 µg/µL stock). Mix immediately. 

SECTION: Day 4    MLi2 or DMSO treatment
5. Treat Cells with MLi2 or DMSO (5uL per well of a 1 mM stock) for120 min, 37 °C 5% CO2

8.   2X rinse with PBS 1x      
SECTION: Fixing and Staining of Cells
6. Fixing: Prewarm freshly made 3% PFA, 4% sucrose in 1X in PBS 1X. You will need 1 mL per well.·
SECTION: Fixing and Staining of Cells
6.1. Aspirate media
9.  2X Wash with PBS1x for 5 min at Room temperature 

SECTION: Fixing and Staining of Cells
6.2. Immediately add prewarmed fixation buffer (3% sucrose, 4 % (v/v) PFA in PBS 1X)·

7. Incubate 12 min Room temperature 

10.  2X Wash with PBS 1x for 5 min at Room temperature ·  
12. Blocking/Permeabilizing: incubate cells blocking/permeabilizing buffer ( 2% BSA + 0.1 % triton X-100 in PBS 1X) for 20 minRoom temperature 

During blocking make and spin the antibody (ab) mix. Each coverslip needs ~40-50 uL uL of ab mix. Antibodies are diluted in Blocking buffer ( 2% BSA in PBS 1X ) and spun at  4 °C 10 min m 50000 x g, 4 °C  to clear aggregates. Remove spun ab and place in new tube. Mix to get even concentration. HERE: we used 1:200 rabbit polyclonal anti-FLAG DYKDDDDK tag – (ptg labs Cat no : 20543-1-AP) 

11. Quenching: 2X rinse and 2x 10 minof 0.4% NH4Cl 75 millimolar (mM) in PBS 1X

SECTION: Day 1: fibronectin coating and cell plating
2.2. Lay one 22 mm x 22 mm glass coverlsip into each 35 mm dish or 6 well. 
22. Find areas with transfected cells. Acquire Z stacks by determining top and bottom with a 0.3 um step size. (about 20-25 z stacks)  ·
23. Analysis in Fiji:  Go through each image, make max projections, and mark each transfected cells with a ROI (region of interest).
24. In an excel sheet keep track of each cell you mark and score as 0 if no LRRK2 filaments are present or 1 if some are.  ·
25. Include at least 50 cells/ sample.  ·
26. Calculate % cells with filaments (number of transfected cells with filaments /total number of transfected cells *100) ·
27. Unblind
28. Transfer values to prism to generate graph and statistics.