TITLE: Transfection by electroporation of GFP-LRRK2 and Immunofluorescent imaging of MEFs VPS35 (D620N) mutants stably expressing LysoTag
AUTHORS: Rotimi Y. Fasimoye, Dario R. Alessi

[DESCRIPTION]
Transfection of foreign DNA and Immunofluorescent (IF) microscopy are powerful tools used in cellular and molecular biology to monitor subcellular localisation of proteins. Mouse embryonic fibroblasts (MEFs) are primary cells notorious for their low transfection efficiency by mostly available chemical methods. This efficiency become even lower if the aim is to express a large sized protein, such as LRRK2, a 250kD protein. Here, we described a method where we used electroporation to transiently transfect GFP-tagged LRRK2 into MEFs. We also used IF microscopy to visualise the subcellular localisation of the transiently expressed GFP-LRRK2. Furthermore, we investigated the colocalization of GFP-LRRK2 with a lysosomal localised TMEM192-3xHA.

[STEPS]
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
1. Place coverslips in 6 well plate (one coverslip per well) and add 2 mL of media. Place the plate in an incubator.

SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
2. Pellet cells from 100% confluent 10cm plate and resuspend in 1 mL media.

SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
3. Count cells and resuspend in media in a way that there are 30000-40000 cells per 10 µL.

SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
4. Power on Electroporator and plug in the electroporator chamber to the output socket.
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
5. Set the following Poring Pulse parameters:
Voltage: 200V
Length: 5ms
Interval: 50ms
Number of cycles: 2
Decay rate: 10%
Polarity: +
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
6. Set Transfer Pulse parameters:

Voltage: 20V
Length: 50ms
Interval: 50ms
Number of cycles: 5
Decay rate: 40%
Polarity: +/-
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
7. Add 3-4 µg of plasmid into a 1.5ml Eppendorf tube.
 

SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
8. Add 90 µL of resuspended cells into the tube. In total, cell number should be approximately 300000-400000. Pipette gently up and down 3 times to mix cell and plasmid.

SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
9. Transfer cell-plasmid mixture into 2mm gap Cuvette and close the cap. Add gently to the side to ensure there are no bubbles. If there are bubbles, tap the cuvette gently on the side until the bubbles move to the top.
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
10. Insert the cuvette into the electroporator chamber and close the lid.
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
11. Briefly measure electrical impedance by pressing the “Ω" button and check reading. This should be between 0.03 and 0.055. 
If reading is too low, add 1-2 µL of cells. 
If too high, add 1-2 µL of media.
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
12. Press start button to begin electroporation process.
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
13. On completion, use Pasteur pipette (provided with the cuvette) to transfer cells into the wells containing a coverslip (from Step 1).
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
14. Rotate plate gently to spread the cells within the well.
SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
15. Return plate into the incubator and incubate for at least 1080 min at 37 °C.

SECTION: Transfection of cells with GFP-LRRK2 plasmid by electroporation
16. Replace media with full Growth media (i.e., media with Pen-Strip) and incubate for another 12-18 hours.
SECTION: Preparing cells for Immunofluorescence imaging
17. Remove media and wash cells.
SECTION: Preparing cells for Immunofluorescence imaging
17.1. Remove media and wash cells with 3 mL PBS +0.2% BSA+0.02% sodium azide for 5 min. (1/3)

SECTION: Preparing cells for Immunofluorescence imaging
17.2. Wash cells with 3 mL PBS +0.2% BSA+0.02% sodium azide for 5 min. (2/3)
SECTION: Preparing cells for Immunofluorescence imaging
17.3. Wash cells with 3 mL PBS +0.2% BSA+0.02% sodium azide for 5 min. (3/3)
SECTION: Preparing cells for Immunofluorescence imaging
18. Fixed cells in 4% w/v PFA. Add 3 mL of dissolved PFA and incubate at Room temperature for 10 min.
SECTION: Preparing cells for Immunofluorescence imaging
19. Permeabilise cells with 1% NP-40 (v/v in PBS +0.2%BSA+0.02% sodium azide).
SECTION: Preparing cells for Immunofluorescence imaging
20. Block with 3% BSA (w/v in PBS) for 30 min.

SECTION: Preparing cells for Immunofluorescence imaging
21. Prepare a combination of primary antibodies (Table 1) as shown below. Antibodies are diluted in PBS +0.2% BSA+0.02% sodium azide.

Rat anti-HA (1:1000) and Chicken anti-GFP (1:1000)
SECTION: Preparing cells for Immunofluorescence imaging
22. Incubate cells at Room temperature with diluted primary antibodies for 60 min. Do this in a humid chamber on a piece of Parafilm. Put a 60 µL drop of diluted antibodies on the parafilm. Carefully place coverslip on the droplet, with the side containing attached cells, facing inward, making contact with the droplet.
SECTION: Preparing cells for Immunofluorescence imaging
23. Wash cells, 3 times, with 3 mL PBS +0.2%BSA+0.02% sodium azide.
SECTION: Preparing cells for Immunofluorescence imaging
24. Prepare a combination of Secondary antibodies as described below (see Table 2 for more information about the secondary antibodies). Antibodies are diluted in PBS +0.2% BSA+0.02% sodium azide.

anti-Rat Alexa 594 (1:500) and anti-Chicken Alexa 488 (1:500).
SECTION: Preparing cells for Immunofluorescence imaging
25. Add 0.5 µL Hoechst 33342 solution for nuclear staining.
SECTION: Preparing cells for Immunofluorescence imaging
26. Incubate cells at Room temperature with diluted secondary antibodies for 60 min. Do this in a humid chamber on a piece of Parafilm. Put a 60 µL drop of diluted antibodies on the parafilm. Carefully place coverslip on the droplet, with the side containing attached cells, facing inward, making contact with the droplet.
SECTION: Preparing cells for Immunofluorescence imaging
27. Wash cells, 3 times, with 3 mL PBS +0.2%BSA+0.02% sodium azide.

SECTION: Preparing cells for Immunofluorescence imaging
28. Rinse cells by dipping briefly in MilliQ water and gently dry on Kleenex wipes.
SECTION: Preparing cells for Immunofluorescence imaging
29. Label microscope glass slides (preferably the one with frosted side) according to the primary antibody used. Take note of the emission wavelength of the probe on the secondary antibodies.
SECTION: Preparing cells for Immunofluorescence imaging
30. Add a drop of VECTASHIELD antifading Mounting media.
SECTION: Preparing cells for Immunofluorescence imaging
31. Mount cover slip (containing cells) on the glass slide, ensuring that the side containing the cells is facing inward, making contact with the oil. Allow to dry for 30 min, ensuring slides are prevented from direct light.
SECTION: Preparing cells for Immunofluorescence imaging
32. Slides can be stored at 4 °C or viewed immediately on a confocal microscope.