Transfection by electroporation of GFP-LRRK2 and Immunofluorescent imaging of MEFs VPS35 (D620N) mutants stably expressing LysoTag
Dario R. Alessi, Rotimi Y. Fasimoye
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Abstract
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.
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Steps
Transfection of cells with GFP-LRRK2 plasmid by electroporation
Place coverslips in 6 well plate (one coverslip per well) and add 2mL of media. Place the plate in an incubator.
Pellet cells from 100% confluent 10cm plate and resuspend in 1mL media.
Count cells and resuspend in media in a way that there are 30000-40000 cells per 10µL.
Power on Electroporator and plug in the electroporator chamber to the output socket.
Set the following Poring Pulse parameters:
- Voltage: 200V
- Length: 5ms
- Interval: 50ms
- Number of cycles: 2
- Decay rate: 10%
- Polarity: +
Set Transfer Pulse parameters:
- Voltage: 20V
- Length: 50ms
- Interval: 50ms
- Number of cycles: 5
- Decay rate: 40%
- Polarity: +/-
Add 3-4µg of plasmid into a 1.5ml Eppendorf tube.
Add 90µL 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.
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.
Insert the cuvette into the electroporator chamber and close the lid.
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
1-2µLof cells. - If too high, add
1-2µLof media.
Press start button to begin electroporation process.
On completion, use Pasteur pipette (provided with the cuvette) to transfer cells into the wells containing a coverslip (from Step 1).
Rotate plate gently to spread the cells within the well.
Return plate into the incubator and incubate for at least 18h 0m 0s at 37°C.
Replace media with full Growth media (i.e., media with Pen-Strip) and incubate for another 12-18 hours.
Preparing cells for Immunofluorescence imaging
Remove media and wash cells.
Remove media and wash cells with 3mL PBS +0.2% BSA+0.02% sodium azide for 0h 5m 0s. (1/3)
Wash cells with 3mL PBS +0.2% BSA+0.02% sodium azide for 0h 5m 0s. (2/3)
Wash cells with 3mL PBS +0.2% BSA+0.02% sodium azide for 0h 5m 0s. (3/3)
Fixed cells in 4% w/v PFA. Add 3mL of dissolved PFA and incubate at Room temperature for 0h 10m 0s.
Permeabilise cells with 1% NP-40 (v/v in PBS +0.2%BSA+0.02% sodium azide).
Block with 3% BSA (w/v in PBS) for 0h 30m 0s.
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)
Incubate cells at Room temperature with diluted primary antibodies for 1h 0m 0s. Do this in a humid chamber on a piece of Parafilm. Put a 60µL 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.
Wash cells, 3 times, with 3mL PBS +0.2%BSA+0.02% sodium azide.
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).
Add 0.5µL Hoechst 33342 solution for nuclear staining.
Incubate cells at Room temperature with diluted secondary antibodies for 1h 0m 0s. Do this in a humid chamber on a piece of Parafilm. Put a 60µL 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.
Wash cells, 3 times, with 3mL PBS +0.2%BSA+0.02% sodium azide.
Rinse cells by dipping briefly in MilliQ water and gently dry on Kleenex wipes.
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.
Add a drop of VECTASHIELD antifading Mounting media.
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 0h 30m 0s, ensuring slides are prevented from direct light.
Slides can be stored at 4°C or viewed immediately on a confocal microscope.
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