Jun 03, 2025
Version 1
Protocol for Creation of Mag-Fura-2 Vesicles for S2E Calcium Flux Assay V.1
This protocol is a draft, published without a DOI.
- 1Miami University
Protocol Citation: Andrew Morris 2025. Protocol for Creation of Mag-Fura-2 Vesicles for S2E Calcium Flux Assay. protocols.io https://protocols.io/view/protocol-for-creation-of-mag-fura-2-vesicles-for-s-gzxgbx7jx
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 23, 2025
Last Modified: June 03, 2025
Protocol Integer ID: 218824
Funders Acknowledgements:
Gary Lorigan
Grant ID: 5R35GM126935-07
Abstract
for creating calcium dye filled liposomes for ratiometric fluorescence-based calcium flux assays with ion channel peptides.
Day 1 - Cleaning Containers
Day 1 - Cleaning Containers
30m
30m
Clean out 3 x 1 L containers for storage of solutions. Use a large container bottle made of polypropylene, rinse it out with 2% Alconox, followed by filling and flushing with 5 volumes of deionized water.
Add 1 full volume of 0.1 Molarity (M) HCl in type 1 water and allow to sit for 00:30:00 . Rinse with 3 volumes of Type 1 water directly from the purification source and allow it to air dry.
30m
Afterwards, one of these bottles can serve as the container for pure type 1 water. Mark it as ‘ultrapure treated water’. Hereafter, only treated pipette tips should come into contact with it and all water for refilling must come directly from the purification source. When refilling, rinse the container with 100 mL of type 1 water and discard before refilling.
Plastic Component Treatment
Plastic Component Treatment
1h
1h
Clean out a large crystallization dish by adding HCl to
0.1 Molarity (M) into the dish filled with type 1 water. Allow to stand for 00:30:00 , then pour out into a waste container. The waste can be neutralized with NaOH and then poured down the drain.
30m
Rinse the vat with type 1 water, then refill it with 10 millimolar (mM) EDTA.
Add plastic items that will be used. Allow these to sit for 00:30:00 , then drain off. Rinse the treated contents 3 times with type 1 water in the dish, pouring off completely each time. These items should include:
- 1 ml, 200 ul, and 20 ul pipette tips
- The black 96 well fluorescence plate and lid
- 3 plastic spatulas
- 2 weigh boats
- 3 x 15 ml falcon tubes
- 10 x 2 ml Eppendorf tubes
- 10 x TLA 120.2 centrifuge tubes
30m
Glass Component Treatment
Glass Component Treatment
30m
30m
Fill the following with 5 % (v/v) nitric acid. Fill completely and allow to sit for 00:30:00 . Pour off the acid into dedicated acid waste, then completely fill and drain the tubes with type 1 water. Repeat this 5x.
- One small glass culture tube per thin film that you will make. Making extra is advisable.
- Glass Hamilton extruder syringes. Pull the dilute acid in by aspiration.
- Glass syringes used for measuring and dispensing chloroform solutions. Pull the dilute acid in by aspiration.
- Several 1.5 dram vials including the inside of their PTFE caps.
Note
Note: if the lipid stock solution has already been prepared and is available it is not necessary to clean new vials for stock storage.
30m
Prepare Reagents and Thin Films
Prepare Reagents and Thin Films
13h 30m
13h 30m
From here forward only use pipette tips that were EDTA treated and rinsed from the previous steps
Buffer A -
- NaCl -1.75 g
- HEPES - 477 mg or 521 mg if HEPES sodium salt
- Trolox - 50.1 mg weighed into one of the cleaned Eppendorf tubes, with 2 mL of DMSO added to solubilize. Once solubilized all of it may be added to the solution.
Regenerate Chelex Resin
Strip any previously bound cations by loading a 10 mL bed of Chelex 100 resin on a gravity flow column. Pass 3 column volumes of 1 Molarity (M) HCl over. The bed volume will shrink, this is normal.
Pass 3 column volumes of type 1 water to rinse.
Regenerate with 3 column volumes of 1 Molarity (M) NaOH, the bed volume will expand back to its original volume.
Pass 5 column volumes of type 1 water over the column.
Incubate the regenerated resin in batch with your prepared buffer for 01:00:00 with nutation, then decant off the buffer from the beads into another cleaned polypropylene bottle.
1h
Adjusting pH
Tare another treated Eppendorf tube and add one flake of NaOH. Weigh it, and add an amount of type 1 water to make 1 Molarity (M) NaOH.
Note
Other stock NaOH used for general pH adjustment should not be used as it may introduce calcium.
Calibrate the pH probe before use using the standards.
Note
Ca binding to the dye is very pH sensitive and we need to be precise, so calibration should be done each time.
Before measuring the solutions, rinse the probe then let it sit in a beaker of 10 millimolar (mM) EDTA for 00:15:00 , followed by brief immersion in type 1 water 3 times.
15m
Adjust the pH to 7.4 with the NaOH.
Calcium Buffer
Transfer 30 mL of your buffer A to one of your EDTA-cleaned falcon tubes. Add 150 µL of 2 Molarity (M) CaCl2.
Before measuring the solutions, rinse the probe then let it sit in a beaker of 10 millimolar (mM) EDTA for 00:15:00 , followed by brief immersion in type 1 water 3 times.
15m
Adjust the pH to 7.4 with the NaOH.
Making Lipid Thin Films
Add 20 nmol worth of peptide in TFE to each cleaned glass tube. Mark each tube for identification based on which site label was used.
Note
- Cysless S2E is 8268.86 g/mol. At 3 mg/mL is 363 micromolar (µM) . 20 nmol equates to 55 µL of stock to be added.
- S2E delta CTD is 4318.26 g/mol. At 3 mg/mL is 695 micromolar (µM) . 20 nmol = 28.8 µL of stock to be added.
- S2E F56C MTSL is at 4.55 mg/mL . 20 nmol = 36 µL of stock to be added.
- Individual labeled cysteine mutants have roughly the same mass which can be assumed to be 8444 Da (including MTSL). This equates to 355 micromolar (µM) when at 3 mg/mL , so 20 nmol = 56.4 µL . Small differences in mass between labeled sites produce differences in volume that are within pipetting error.
For 1 : 250 peptide : lipid ratio, 50 µL of 100 millimolar (mM) 3:1 POPC:POPG in chloroform should be added to each. For dispensing solutions in chloroform, glass syringes which have been acid washed should be used. One additional tube, to which no peptide has been added should also receive lipid. This will serve as a blank negative control.
Dry the thin films using nitrogen gas stream to create a vortex that spreads it out evenly along the walls.
Place the dried thin films in a vacuum desiccator overnight and turn on the vacuum.
12h
Day 2 - Dye Buffer
Day 2 - Dye Buffer
Put 30 µL of aqueous stock at 1 millimolar (mM) into 6 mL of Buffer A (200 x dilution) to make 5 micromolar (µM) dye. Measure and store in an EDTA-cleaned tube.
Test that calcium contamination is acceptably low.
Take 90 µL of dye buffer and dispense into each of 4 wells on the EDTA-washed 96 well plate, in each well add 10 µL of either
0 micromolar (µM) , 30 micromolar (µM) , 100 micromolar (µM) , or
500 micromolar (µM) EDTA solution to produce 10 fold dilutions.
Mix the samples on a plate shaker. If available, this can be done inside the plate reader.
96_well_exitation_scan.prt16KB Measure the excitation spectrum by opening the protocol 96_well_excitation_scan.prt in the Gen5 application on the plate reader computer. the protocol file is attached.
The excitation scan protocol must be changed manually each time within the program to correctly select the wells used for scaling the gain on the PMTs to whichever wells you put your test samples in. See the screenshots below. click the areas highlighted.
Expected result
Excitation spectra should ideally show that Addition of EDTA to concentrations higher than 3 micromolar (µM) does not change the excitation spectrum or shift the ratio of 340 nm to 380 nm compared to lower levels of added EDTA. Since mag-fura-2 has KD of about 40 uM towards Ca, low levels of trace calcium below 3 micromolar (µM) , as determined by the minimum amount of EDTA required to produce a 0 baseline (see Fig 3A of doi: 10.1152/ajpcell.1989.256.3.C540) will be small enough to have negligible impact on the assay. Larger amounts of calcium indicate contamination, and solutions should be remade and glassware and plastics cleaned more thoroughly.
Vesicle Formation
Vesicle Formation
Add 500 µL of dye buffer to each thin film, gently mix to avoid foaming from the Trolox, and freeze thaw at least 3 times.
Ensure that the sides of the tube where lipid film is present is washed this way and subject to freezing by swirling the tube gently in a circle when freezing in liquid nitrogen. Repeat this up to 9x to homogenize further if back-pressure on the syringes is very high when extruding. Note that you do not need to let tubes come into thermal equilibrium with the liquid nitrogen, doing so will only make it take longer for them to thaw. They simply need to get cold enough to freeze solid.
Prepare the extruder according to normal assembly protocol, use a 100 nm membrane. Once assembled, draw some 10 millimolar (mM) EDTA solution into a syringe and push through the extruder. Repeat in the reverse extrusion direction, then discard the solution. Fill one syringe completely with type 1 water and extrude it into the other syringe, then discard the water. Repeat this wash in the reverse direction, alternating each way for 5 total water washes to remove all trace calcium and EDTA. Then equilibrate the device into Buffer A by passing a small amount over, and discarding the buffer.
For each mutant, aspirate it from the tube into a cleaned Hamilton syringe and push out any air. Extrude each in 21 passes.
Note
If the number of passes changes, always make sure it is an odd number so that you end with the liquid in the syringe that you didn't start with. this ensures large, clogging particles that may have been present initially are not in the extruded sample.
Dispense the final extruded liposomes into an EDTA-washed TLA 120.2 centrifuge tube.
Between each mutant clean the syringes and extruder. Aspirate 200 µL of Buffer A into each syringe and eject it to rinse. Then, aspirate 200 µL of buffer A into a syringe and pass across the extruder. Repeat this process 4 times, alternating directions each time.
Pellet liposomes by ultracentrifugation of the tubes with collected samples at117000 rpm, 4°C , TLA 120.2 rotor. 600000 RCF . Balance tubes by addition of necessary Buffer A
Pour off the supernatant of each centrifuged tube. The pellet should be very firm and not be affected. Pull buffer away by capillary action by placing the tube upside down on a paper towel.
Resuspend each pellet in 1 mL of Buffer A, aspirating and dispensing directly onto the pellet to break it up and resuspend it thoroughly.
Pellet liposomes by ultracentrifugation of the tubes with collected samples. 117000 rpm, 4°C , TLA 120.2 rotor. 600000 RCF balance tubes by addition of necessary Buffer A.
Pour off the supernatant of each centrifuged tube. The pellet should be very firm and not be affected. Pull buffer away by capillary action by placing the tube upside down on a paper towel.
Resuspend each pellet in 200 µL of Buffer A, aspirating and dispensing directly onto the pellet to break it up and resuspend it thoroughly.
Note
Try to avoid excessive frothing when resuspending by pipetting.
If moving immediately to assay, the resuspended liposomes can be aspirated and transferred to to 96 well EDTA-washed plate, otherwise they should be dispensed into EDTA-treated eppendorf tubes from Step 5 and then stored at 4 °C
Prepare sealed 96 well plate
Prepare sealed 96 well plate
Apply a uniform layer of silicone grease ~2 mm thick to the bottom of a small, clean glass beaker
Note
If your plate reader timecourse assay runs for less than ~2 hours, these steps are not necessary. The purpose is purely to prevent evaporative loss from wells over long timecourses that changes the concentration of the well contents over time.
use the beaker as a stamp to evenly apply silicone grease to the rim of the wells on a 96 well plate. This is a separate plate from the one used for the assay and is just used to neatly transfer grease in a ring.
evenly place the lid over the template plate and transfer the silicone grease. press sufficiently to be able to see close ring seal around your wells that you will be using in the assay.
remove the lid carefully and inspect that there is a grease ring arround each well. the lid will be used for our EDTA cleaned well from earlier.
Note
It is critical that the grease forms a complete seal so that water vapor cannot escape when the lid is placed on. it is also critical however that no grease obstructs the window in the center of the lid above each well.
Timecourse Assay
Timecourse Assay
12h 30m
12h 30m
Dispense 198 µL from each liposome sample produced into its own well in the EDTA cleaned 96 well plate.
Note
It is advisable to avoid wells in columns 1 and 12, as they have a tendency to not form tight grease seals with the plastic lid
Place the greased lid over the plate to cover it, press down enough to form a seal over the wells you are using.
Note
When handling the lid, ensure you do not leave fingerprints or smudges on the outside. The reader needs an undistorted optical path into the well. If you need, wipe down the outside of the lid with ethanol and a kimwipe.
Place the plate in the reader with column 1 oriented towards the reader, before closing the tray, ensure the Use lid checkbox is checked, otherwise the plate reader will jam up and eject the plate.
96 well fura2 timecourse 12 hr.prt92KB Open the Gen5 software and select the protocol
96 well fura2 timecourse 12 hr.prt
attached here.
Click the button in the menu bar to start the assay.
The plate will read for an initial excitation spectrum, then a 15 minute timecourse to average baseline excitation ratios at 340 and 380 nm. After this it will eject the plate and prompt to add calcium solution. Add 2 µL of the previously made calcium buffer to each well, then click 'okay' on the dialog box.
Note
Effort should be made to minimize the time spent pipetting. Be fast and efficient.
The assay will now run for 12:30:00 . At the end, the user is instructed to save an excel file containing the both raw and processed data.
12h 30m
after the 12 hrs. the plate will be ejected. remove the lid and add 4 µL of 1.02 mM melittin peptide in water to each well. Add the lid back and gently press the seal. click okay and the instrument will take the plate back in. It will then shake for 1 minute and let incubtate for 10 minutes. 15 minutes of timecourse 340/380 ratio measurements are then taken.