Immunofluorescence-based assay to assess LRRK2 association with microtubules in HEK293 cells

Prepare a transfection mix by adding 0.6µg of Flag-LRRK2 (or Flag-empty vector) cDNA and 1.8µL of 1mg/mL PEI solution into 150µL of Opti-MEM for each well. Vortex for 0h 0m 20s/0h 0m 30s.

Incubate the transfection mix for 0h 20m 0s at Room temperature to allow the DNA/PEI complex to form.

For each LRRK2 construct, add 150µL of the transfection mix to each well.

Remove culture medium from one flask of HEK293 cells.

Briefly rinse the cell layer with 0.25% (w/v) Trypsin - 0.53millimolar (mM) EDTA solution to remove all traces of serum.

Add 2mL of Trypsin-EDTA solution to the flask and incubate at 37°C until the cell layer is dispersed.

Add 8mL of complete growth medium and resuspend cells by gently pipetting.

Count the cells using the method of choice.

Resuspend the cells to a concentration of 8x10⁴ cells per ml of complete growth medium.

Add 1mL of cell suspension (8x10⁴ cells) into the well containing the transfection mix.

Transfer the plates to a humidified incubator maintaining 37°C and 5% (v/v) CO₂.

45 hours after transfection, treat cells with 100nanomolar (nM) MLI-2 or 0.1% (v/v) DMSO (control vehicle) and incubate for 3h 0m 0s at 37°C in a humidified incubator maintaining 5% (v/v) CO₂.

After treatment with MLi-2/DMSO, remove culture medium completely from each well using an aspirator.

Immediately add 50µL of ice-cold complete lysis buffer to each well ensuring that the entire surface is covered by lysis buffer.

Transfer the plate 37On ice.

Scrape the cells on the dish using a cell lifter to ensure all cells are detached from the well.

Using a pipette, transfer the lysate to a 1.5 mL Eppendorf tube.

Leave samples 37On ice for 20/30 minutes to allow for efficient lysis.

Spin down lysates at 17000x g,4°C.

Transfer supernatant to a new Eppendorf tube and discard the pellet.

Proceed to quantitative immunoblotting analysis as described in dx.doi.org/10.17504/protocols.io.bsgrnbv6 (Quantitative Immunoblotting Analysis of LRRK2 Signalling Pathway).

45 hours after transfection, treat cells with 100nanomolar (nM) MLI-2 or 0.1% (v/v) DMSO (control vehicle) and incubate for 3h 0m 0s at 37°C in a humidified incubator maintaining 5% (v/v) CO₂.

After treatment with MLi-2/DMSO, remove culture medium completely from each well using an aspirator.

Fix cells by adding 4% (v/v) PFA in PBS pre-warmed to 37°C.

Incubate for 0h 10m 0s at 37Room temperature.

Remove PFA completely using a pipette and wash with PBS.

Permeabilise cells by incubating with 1% (v/v) NP-40 alternative in PBS for 0h 10m 0s at 37Room temperature.

Remove the solution completely using an aspirator.

Block with 1% (w/v) BSA in PBS for 1h 0m 0s at 37Room temperature.

Prepare the primary antibody solution by diluting anti-Flag M2 antibody and anti-β-tubulin antibody in 0.2% (w/v) BSA in PBS (1:1000 and 1:500 dilution, respectively).

Incubate the samples with primary antibodies for 2h 0m 0s at 37°C in a humidified chamber.

Wash the samples 3 times with 0.2% (w/v) BSA in PBS (10 minutes per wash).

Prepare the secondary antibody solution by diluting the secondary antibodies (Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit) in 0.2% BSA in PBS (1:500 dilution). Add DAPI at 1 final concentration to the secondary antibody solution.

Incubate the samples with the secondary antibodies and DAPI at 37Room temperature for 1h 0m 0s in the dark.

Wash the samples 3 times with 0.2% (w/v) BSA in PBS (10 minutes per wash).

Image cells using a Zeiss LSM 710 or 880 laser scanning microscopes using the x40 EC Plan-Neofluar (NA 1.3) objective with a zoom of 0.6 and optical section thickness of 1.0 mm (image size 2048x2048 pixels, pixel size 0.173 μm).

Image 50-100 cells from 4-6 randomly selected fields with Alexa Fluor 488-positive cells for each well (These are cells successfully transfected with Flag-LRRK2).

Perform cell counting using the Image J Cell Counter feature.

Once cell counting is done, data is unblinded and analysed as percentage of cells for each category using GraphPad Prism and applying a 2-way ANOVA and the post-hoc Dunnett’s test to evaluate statistical significance between different experimental conditions.

Copy the code below and change the folder directories according to where the data is located and where you would like the data with changed names to be located (the original data will not be changed).

import os
import string
import random
import shutil

def random_name(size=9, chars=string.ascii_uppercase + string.digits): 

#This defines the random name as a string of 9 letters and digits.

return ''.join(random.choice(chars) for _ in range(size))

list=os.listdir("C:/Documents/Folder with the data/”) 

#This is the folder containing the images.

for k in list: decode=open("decode.txt", "a") 

#This creates a log of the original file names and new randomised file names so these can be decoded later.

codename=random_name()
print(codename)
decode.write(codename+".lsm"+'*'+k+'\n') 

#Note: Change “.lsm” to whatever format you are using for the image files.

shutil.copy("C:/Documents/Folder with the data/"+k, "C:/Documents/Folder for the renamed data/"+codename+".lsm") 

#The first folder is the folder containing the original image files (as before) and the second one is the folder for the renamed images to be copied to.

Select “Run” and the program will create the renamed copies for you to use.