TITLE: Preparation of cells for live-cell imaging of phosphoinositide reporters by total internal reflection fluorescence (TIRF) microscopy
AUTHORS: Ralitsa R Madsen

[DESCRIPTION]
This protocol provides detailed instructions for transient expression of PIP3/PI(3,4)P2-selective PH domain-based reporters for subsequent live-cell imaging by total internal reflection fluorescence microscopy. Instructions are also provided for execution of perturbation experiments using growth factors or inhibitors that act through the PI3K signalling pathway. The protocol was originally developed with advice from Dr York Posor (Vanhaesebroeck Lab, University College London), Dr James Burchfield (University of Sydney) and Dr Alison Kearney (David James Lab, University of Sydney).

The protocol is routinely used in our lab for experiments with HeLa cells. It has also been used with mouse embryonic fibroblasts and lung adenocarcinoma A549 cells, however the complete medium (CM), seeding densities and transfection conditions differ for each cell line.  

We culture our cells without antibiotics except during live-cell imaging as indicated.

For subsequent data analysis, please refer to the pipeline provided on the Open Science Framework under doi: 10.17605/OSF.IO/4F69N.

[BEFORE_START]
Prepare coating solution: thaw Matrigel stock aliquot on ice, then dissolve 400 µl in 19.6 ml of cold OptiMEM 
Prepare autoclaved forceps
Prepare complete medium (CM) in advance, e.g. for HeLa cells (see materials for individual items): DMEM with L-Glutamine and Sodium Pyruvate (500 ml) + L-Glutamine (5 ml) + FBS (50 ml)
Prepare live cell imaging (LCI) medium: 49 ml Fluorobrite DMEM + 500 µl L-Glutamine + 500 µl Penicillin-Streptomycin 
Make sure that the microscope, including objectives, holders and oil are warmed up to 37 degrees Celsius for least 3 hours prior to set up for imaging
Assemble the fluidics system by threading the end of a 40-50-cm-long piece of PFTE tubing into a 1.5-2 cm-long piece of Pharmed tubing with a luer lock at the other end. Roll the tubing and store away in a 150 cm putri dish when not in use. When in use, the end of the PFTE tubing that is free must be threaded through an appropriately sized hole in the lid of an imaging dish - this hole can be made using a flame to heat a needle, then melting the plastic in the lid until the required size has been reached.

[GUIDELINES]
Use filter tips for all cell culture and molecular biology workflows. 
Ensure that cells are healthy and have been passaged at least once since thawing prior to using them for experimentation
Ensure that all solutions are pre-warmed to room temperature before they come in contact with cells
Ensure that all plasmids used for transfection are of high purity and without endotoxins (we advise additional validation by restriction enzyme digests and Sanger sequencing after plasmid prepping)
We recommend making 50 ml aliquots of stock media solutions (e.g., complete medium and live-cell imaging); if kept in the fridge, we use these for up to 1 month. 
Some cell lines (e.g., human pluripotent stem cells) may not grow well in the glass bottom Ibidi dishes even if these are coated; we would instead recommend the use of the equivalent dishes with Ibidi polymer coverslips with IbiTreat (Ibidi #81156)

[STEPS]
SECTION: Dish assembly and coating
1. Take the required number of Ibidi dishes that will be needed for the experiment, along with the equivalent number of inserts. (NB: you can reuse inserts once provided that they have been sterilised in 70% ethanol and dried since the last use; we do this by passing them through PBS once, then 2x across two distinct 70% ethanol aliquots in 50 ml Falcons, then transfer to dry in a closed Petri dish).  

SECTION: Dish assembly and coating
2. Using ice-col Matrigel solution (1:50 dilution in OptiMEM, prepared in advance), coat each well with 100 µL. 

SECTION: Dish assembly and coating
2.1. 5 mL Note the coating time and transfer the dishes to the incubator for a minimum of 45 min (NB: we usually seed within 1-2h of coating). 

SECTION: Dish assembly and coating
1.1. Using autoclaved forceps, pick up the individual inserts and place an insert in each dish. Invert the dish once to check that the insert is sticking to the bottom of the dish.
SECTION: Dish assembly and coating
1.2. Label the lid of the dish with the well layout. To ensure that you can keep track of the orientation even when you remove the lid, label both lid and base with "N" (north) and "S" (south) in the equivalent positions (see attached schematic).  
 

SECTION: Cell seeding (the given volumes for splitting are for HeLa cells in T25 flasks)
3. Take the cells for processing out of the incubator and confirm healthy state by light microscopy examination. The cells should be 80-90% confluent.
4. Remove the spent medium and wash the cells with 5 mL  DPBS.    

SECTION: Dish assembly and coating
2.2. Make sure to keep the Matrigel solution on ice and return to the fridge when done. 
5. Remove the DPBS and add 0.75 mL TrypLE Express. 

6. Incubate the cells for 8 min at 37 degrees Celsius.

6.1. If you are processing more than one cell line (e.g. different HeLa mutants), proceed with the next, ensuring that you stagger individual cultures 2 minutes apart. DO NOT process multiple cell lines for splitting the same time as this poses a cross-contamination risk. 
7. Once the cells have dissociated, add3.3 mL of complete medium (CM), resuspend the cells and transfer to an appropriate labelled 5 ml Eppendorf tube. The final volume should be approximately 4 ml.

7.1. Immediately after transfer, mix 20 µL of the cell suspension with 20 µLTrypan Blue. 

8. Count the cells using a haemocytometer (NB: we find this method to be the most reliable compared to automated cell counting)
9. Dilute the cell suspension to 30000 cells/ml (NB: ensure sufficient mixing at all stages, including of the stock cell suspension) in a final volume of 1 mL (use 1.5 ml Eppendorf tubes).

10. Take out the coated dishes from the incubator.
10.1. For each well to be seeded, take the respective cell suspension and mix 2-3 times with a P1000 Gilson pipette set at 500 µl. 
10.2. Immediately after mixing the cell suspension, aspirate the coating solution from the well to receive cells using non-filter tips (be careful not to let the wells dry out, yet also take care to observe sterile techniques!).
10.3. Transfer 100 µL (3000 cells) of the mixed cell suspension to each well. Make sure not to tilt the dish at any point after cell seeding. (NB: if you left the mixed cell suspension sit for more than 1-2 minutes, mix again before seeding though be mindful of time as the wells dry out once the coating has been removed).

10.4. Repeat this procedure for the remaining cell suspensions.
10.5. Once you have seeded all wells, leave the dishes in the tissue culture cabinet for 15-20 minutes before transfer to the cell culture incubator. This ensures a more even seeding distribution. 
11. Leave the cells in the cell culture incubator for 20-28 hours after seeding.
SECTION: Reporter plasmid transfection
12. Pre-equilibrate FuGENE to room temperature and vortex vigorously before use.
SECTION: Reporter plasmid transfection
13. Prepare the transfection mastermix as follows (example given for 8 transfections + 2 extra)
 

SECTION: Reporter plasmid transfection
13.1. Immediately after adding FuGENE, mix the suspension by pipetting multiple times, then leave to incubate for 10-15 minutes.
SECTION: Reporter plasmid transfection
14. After incubation, mix the transfection mix 1:10 with complete medium (e.g. mix 100 µl transfection mix with 1000 µl complete medium, for a final volume of 1100 µl)
SECTION: Reporter plasmid transfection
15. Working with maximum two wells at a time, aspirate the spent medium solution from the cells using non-filter tips and quickly albeit gently, add 110 µL transfection mix to each well, taking care not to touch the cell monolayer 
NB: change filter tips in between each addition to minimise cross-contamination risk.
NB: if you are too slow at this stage, the cells will not transfect properly as the cell monolayers dry very quickly once the medium is removed from the small wells. 

SECTION: Reporter plasmid transfection
13.2. Five minutes before the transfection mix incubation is complete, take the seeded cells out of the incubator and inspect under the light microscope to check cell health, then transfer to the tissue culture cabinet.
SECTION: Reporter plasmid transfection
16. Return the transfected cells to the incubator for 20-28 hours.
SECTION: Serum-starvation
17. Approximately 24 hours following transfection, inspect the cells under the light microscope to check health, then transfer to the tissue culture cabinet.
SECTION: Serum-starvation
18. Using autoclaved forceps, remove the insert from each dish (NB: either discard if it has been used once before, or sterilise as described in Step 1).
SECTION: Serum-starvation
19. Using non-filter tips, aspirate the spent medium from the cells from the area where the insert boundary is visible (do not touch the cells). 
SECTION: Serum-starvation
20. Gently add 3 mL of live cell imaging (LCI) medium to the dish, taking care to cover the inner circle containing the cells. This is a wash step to ensure phenol red and serum removal.

SECTION: Serum-starvation
21. Remove the LCI wash and replenish the cells with exactly 2 mL LCI, then return to the incubator for 120 min prior to microscopy set-up. 

SECTION: Preparation of treatment solutions
22. During the 2 hour wait, prepare the treatment solutions in LCI following the example tables below for 100 nM final stimulations with IGF1 and EGF (note that the solutions are prepared at a higher concentration for final dilution to the required 1X amount once added to the cells in volumes of 500 µl). Make sure to mix all stock solutions prior to final pipetting. 


 


 




SECTION: Live-cell imaging and treatment additions
23. Following 2 hours of serum-starvation, bring the cells on the microscope and proceed with setting up which takes around 45 min to 1 (NB: you can only work with 1 dish at a time, therefore make sure that cells in different dishes have not had serum removed at the same time)
SECTION: Live-cell imaging and treatment additions
24. Connect the fluidics system to the dish by exchange the lids. Make sure that the fluidics system is secured (use white Blu-Tack only) and the dish is clamped in place. Ensure stable CO2 and temperature conditions.
 
 

SECTION: Serum-starvation
21.1. When imaging, you can only work with one dish at the same time for fast acquisition, therefore make sure that you do not serum-starve the cells in multiple dishes at the same time. Instead, stagger starvation timings to ensure that cultures are not starved for more than 3 hours at the time of imaging start (2 hours in incubator + 1 hour at the microscope while setting up)
SECTION: Live-cell imaging and treatment additions
25. Set-up the microscope with the correct laser and exposure settings (these will be microscope-specific, but we advise careful checking of the TIRF angle to ensure that the plasma membrane is in focus, with minimal signal from the cytoplasm)
SECTION: Live-cell imaging and treatment additions
26. Select individual cells for imaging and specify positions, making sure that a cell expresses both EGFP and mCherry, i.e. both wildtype and mutant PH domain reporter.
SECTION: Live-cell imaging and treatment additions
26.1. Make sure to only select cells that do not have a saturated signal, yet have a signal that is at least twice as strong as the surrounding background. Be mindful of the fact that the signal is weaker with the mutant mCherry-tagged PH domain reporter. 
SECTION: Live-cell imaging and treatment additions
27. Specify the number of time points: we usually image every 70 seconds for 60 minutes, across a total of 28 positions, with a 60X or 100X TIRF oil objective.
SECTION: Live-cell imaging and treatment additions
29. Once imaging has completed, clean the fluidics system, clear the microscope out and proceed with data analysis.
SECTION: Live-cell imaging and treatment additions
29.1. To clean the tubings after each experiment, first flush with 3-4 ml 70 % ethanol (using syringe), then 3-4 ml MilliQ water, then push air through 3-4 times and leave to dry.
SECTION: Serum-starvation
21.2. Remember to switch on the microscope temperature unit on at 37  °C at least 2h in advance of initiating imaging, and ensure that all components (including lens and dish holders) are heated up.
SECTION: Live-cell imaging and treatment additions
28. Start imaging and proceed with treatments at the required time points. We usually attach the syringes to the fluidics system in advance and 30s before treatment needs to commence, we pipette the solutions in each syringe, then push it through at the required time point, ensuring that we mix gently 3-4 times by performing several strokes of up to 0.5 ml with the syringe.

NB: Be sure to check on CO2 levels throughout imaging. The cells and their signalling responses are very sensitive to fluctuations in CO2 and pH. This is a common reason for inconsistent result generation.