Calcein (Fluorexon)

This protocol uses the Calcein (Fluorexon) leakage test to test the membrane integrity of ion carriers^[4] as an example. It only provides experimental guidance and actual application must be modified according to your actual needs.

Ⅰ.Materials Required：

1. Buffer A (200mL)

a. Mix 4mL of 0.5M HEPES pH 7.4 (final 10mM) with 60mL of 0.5M KCl (final 150mM).

b. Add 100mL of deionized water and adjust pH with KOH to 7.4.

c. Top up to a final volume of 200mL with deionized water, and filter-sterilize with a 0.22µm filter.

2. Calcein (Fluorexon) stock in buffer A (16.6mM, 1mL)

a. Dissolve 10.334mg Calcein (Fluorexon) to a final volume of 1mL in buffer A by vortexing.

b. Cover the tube with aluminum foil to protect it from daylight.

3. CuCl₂ stock (2mM)

a. Dissolve 110.98mg CuCl₂ to a final volume of 1mL in deionized water, to obtain1M CuCl₂.

b. Then, add 2µL of 1M CuCl₂ into 998µL of deionized water.

4. G50-Gel in buffer A

a. Add 2.5g Sephadex G-50 to 50mL of buffer A, and dissolve it by mixing.

b. The gel has to swell at RT ON, and is then stored at 4℃.

5. HEPES, pH 7.4 (0.5M, 200mL)

a. Dissolve 9.532g HEPES to a final volume of 170mL in deionized water.

b. Adjust the pH with 1M KOH to 7.4, and top it up to 200mL with deionized water.

6. Ionophore stock (6mM)

a. Prepare an initial stock of 10mg 4-Br-A23187 in 1mL of DMSO.

b. To reach 6mM 4-Br-A23187, 36.15µL of the stock solution is diluted with 63.85µL of filter-sterilized DMSO, resulting in a final volume of 100µL.

c. Before the experiments, the DMSO is filter-sterilized using a 0.22μM syringe filter system.

7. KCl (0.5M, 200mL)

a. Dissolve 7.455g KCl to a final volume of 200mL in deionized water.

8. KOH (1M)

a. Dissolve 5.611g of KOH to a final volume of 100mL in deionized water.

9. Lipid stock in chloroform

a. Lipids are ordered in chloroform at a concentration of 25mg/mL, and stored at -20℃until further use. For longer storage, evaporate the chloroform and store the dried lipid at -20℃. Before using it, dissolve the 25mg lipid in 1mL of chloroform:methanol solution (v:v).

Note: Some lipids may have limited or very poor solubility in chloroform:methanol, and require a mixture of chloroform:methanol:water.

10. Loading buffer (1mL)

a. Take 18.07µL of 16.6mM Calcein (Fluorexon) stock (final 300µM) and top up with buffer A to a final volume of 1mL. Cover the tube with aluminum foil.

11. Triton 100-X solution (20%w/v)

a. Dissolve Triton 200mg in 1mL of deionized water.

Ⅱ. Experimental Procedure

A.Preparation of the lipid film

1. Clean the Hamilton syringes by flushing them five times with chloroform:methanol (1:1, volume/volume) under a fume hood.

Note: Chloroform is a hazardous solvent. Conduct all work in a fume hood, while wearing appropriate personal protective equipment.

1. Using Hamilton syringes, transfer 200µL of a 25mg/mL^-DOPC stock solution (see Recipe 9) into a round bottom glass tube placed on ice.

Note: Avoid any use of plasticware when handling organic solvents.

1. Evaporate the organic solvent at room temperature (RT) under in a rotary evaporator at the reduced pressure of 250mbar overnight, followed by evaporation at~10mbar for 15min, see Figure 1.
2. The dried lipid film can be stored at -20℃.

Figure 1. Scheme of lipid film preparation.(A) The desired amount of lipid (I) in chloroform is transferred to a glass tube. Subsequently, the solvent is evaporated in a rotary evaporator under the reduced pressure of 250mbar overnight (ON), resulting in a thin lipid film on the sides of the glass tube (II). (B) The glass tube containing the desired lipids in chloroform is connected to the rotary evaporator.

B. Preparation of Calcein (Fluorexon)-loaded large unilamellar vesicles (LUVs)

1. Add 1mL of loading buffer (see Recipe 10) and a 3mm glass bead to the lipid film. Vortex for 10min.

2. Transfer the lipid suspension to a new glass tube, without the glass bead.

3. Subject the lipid suspension to five freeze-thaw cycles by placing the tube alternatively into liquid nitrogen for 10min for freezing and in a water bath at 50℃ for 5min for thawing.

Note: Wear a face shield and insulating gloves when handling liquid nitrogen. Use glass tubes with high thermal shock resistance.

4. Assemble the mini-extruder, consisting of two Hamilton glass syringes, a Teflon cylinder with two drain discs, and a 200nm polycarbonate membrane in between them, see Figure 2.

Note: We routinely use polycarbonate membranes of 200nm pore size and perform the extrusion at RT. However, for lipids containing saturated, long-chain fatty acids, which have a high lipid phase transition temperature (T_m), the extruder needs to be heated up to a temperature 5–10℃ above the T_m. This temperature can vary greatly between lipid formulation.

Figure 2. Scheme for preparation of Calcein (Fluorexon)-loaded LUVs. (A) The dry lipid film is hydrated in a loading buffer containing the fluorescent dye Calcein (Fluorexon) (stepI) by vortexing for 10min (stepII). The lipid suspension (stepIII) is subjected to five freeze-thaw cycles using liquid nitrogen (-196℃, stepIV) and a water bath at 50℃ (stepV). Next, the suspension is passed 11 times through 0.2μm nucleopore polycarbonate membranes mounted in a mini-extruder to form LUVs (stepVI). (B) Assembly of mini-extruder. Use four filter supports, two on each side. Use two polycarbonate membranes, with blank sides towards each other.

5. Check the tightness of the mini-extruder by flushing with 1mL of buffer A (see Recipe 1), i.e., pass back and forth between the syringes three to four times. Continue only if the buffer volume stays the same for each passing.

6. Fill one Hamilton glass syringe with ~1mL of lipid/ Calcein (Fluorexon) solution, and place the filled syringe into one end of the mini-extruder.

7. Carefully place an empty syringe into the opposite end of the mini-extruder. The plunger of the empty syringe should be depressed completely into the syringe barrel.

8. Extrude lipid suspension by passing through the filters a minimum of 11 times, starting in syringe 1 and finishing in syringe 2.

Note: The number of passages through the extruder needs to be uneven so that the final liposome sample is collected in syringe 2, which is uncontaminated by residual multilamellar vesicles that have never passed the extruder.

9. Inject the final lipid solution into a 1.5mL microcentrifuge tube, and store it at 4℃.

Note: Size distribution of the resulting liposomal preparations can be evaluated by dynamic light scattering. When using polycarbonate membranes of 200nm pore size, we typically obtain a preparation with an hydrodynamic diameter in the range of 168.5 ± 2.5nm.

10. Immediately wash all parts of the extruders with Milli-Q water, then with 70% ethanol, and dry it thoroughly before storing. Solvent-rinse syringes before storing. When extruding additional vesicles, disassemble and clean all parts of the extruder, and replace the membrane and filter supports.

C. Separation of Calcein (Fluorexon)-loaded LUVs from free dye

1. Prepare two G50 columns, using 2mL plastic syringes without a plunger, and filter supports as a stopper, see Figure 3.

2. Place the syringes into disposable 15mL reaction tubes, add 3mL of Sephadex G50 fine slurry (see Recipe 4) using a Pasteur pipette, centrifuge at 180×g for 5min, and transfer the columns to a new 15mL reaction tube.

3. Load the sample on the top of one of the G50 columns.

4. Centrifuge at 180×g and RT for 5min (see Figure 3).

5. Transfer the eluate to the top of the second G50 column.

6. Centrifuge again at 180×g and RT for 5min (see Figure 3C).

Note: Repeat the size exclusion chromatography of the eluate with a new G50 column, if you do not achieve a separation of the free dye (intense yellow part of the column) from the vesicles (colorless lower part of the column).

7. Collect the eluate containing the Calcein (Fluorexon)-loaded LUVs in a new 15mL Falcon tube.

8. Cover the tube with aluminum foil.

Note: Calcein (Fluorexon)-loaded LUVs could be stored at 4℃ in the dark until the next day.

Figure 3. Separation of Calcein (Fluorexon)-loaded LUVs by size exclusion chromatography. (A) For size exclusion chromatography, two filter supports are placed as a stopper in a 3-mL plastic syringe without a plunger, and 3 mL of Sephadex G50 fine slurry is added. (B) Calcein (Fluorexon)-loaded LUVs are separated from free Calcein (Fluorexon) during the size-exclusion chromatography. The LUVs will elute with the void volume in the early fractions, whereas the non-liposome-associated Calcein (Fluorexon) will elute in later fractions. (C) Image showing a G50 filtration column after the filtration with the trapped free Calcein (Fluorexon). Free Calcein (Fluorexon) is in the first G50 filtration column after the first separation. After the next separation, the second G50 filtration column contains no free Calcein (Fluorexon).

D. Monitoring ion permeability

1. Cool buffer A (see Recipe 1), the cuvette, and the Calcein (Fluorexon)-loaded LUV at 10℃ (e.g., by storing in a fridge at 10℃).

Note: To suppress the rate of uncatalyzed transport in the absence of the ionophore, the assay is performed at 10℃. Liposomes of other lipid compositions might exhibit lower passive permeability and thus allow higher assay temperatures.

2. Turn on the fluorometer and set up the parameters as follows: excitation wavelength 480nm, emission wavelength 520nm, and measurement duration approximately 10min, with 1s resolution. Adjust slits as necessary; a bandpass of 3nm is usually sufficient.

3. Cool down the sample holder of the fluorometer to 10℃.

Note: We use Peltier-based temperature control with magnetic stirring, providing temperature stability and full control software during the measurements.

4. Add 20µL of Calcein (Fluorexon)-loaded LUV in the fluorometer cuvette, and top up to 2mL using precooled buffer.

a. For probing the impact of ionophores on ion permeability, add 1µL of ionophore (see Recipe 6, final concentration 3µM) into the cuvette, and incubate in the fridge for a further 5min.

b. As a control for quenching of Calcein (Fluorexon) in the presence of ions, prepare one sample by adding 10–20µL of 20% Triton X-100 (see Recipe 11) into a cuvette, and incubate in the fridge for 5min.

5. Introduce the cuvette into the fluorometer (remember to include the magnetic stir bar), and start monitoring the emission intensity.

6. Wait until fluorescence is stable (90s).

7. Measure the fluorescence intensity. After 1.5min, add 3µM CuCl₂(3µL of 2mM CuCl_2,see Recipe 3), and record the fluorescence for another 8.5min.

Note: To ensure your setting and the Calcein (Fluorexon)-loaded LUVs are working properly, run an excitation scan from 450 to 500nm (emission 520nm) and an emission scan from 500 to 550nm (excitation 480nm) at the start of the measurements. For Calcein (Fluorexon), the excitation and emission maxima should be at 480nm and 520nm, respectively.

8. The instrument exports the data for later analysis.

References:

[1]. Allen T M, Cleland L G. Serum-induced leakage of liposome contents[J]. Biochimica et biophysica acta (BBA)-biomembranes, 1980, 597(2): 418-426.

[2]. Tsao Y S, Huang L. Sendai virus induced leakage of liposomes containing gangliosides[J]. Biochemistry, 1985, 24(5): 1092-1098.

[3]. Patel H, Tscheka C, Heerklotz H. Characterizing vesicle leakage by fluorescence lifetime measurements[J]. Soft Matter, 2009, 5(15): 2849-2851.

[4]. Uzun H D, Vázquez-Hernández M, Bandow J E, et al. In vitro Assay to Evaluate Cation Transport of Ionophores[J]. Bio-protocol, 2022, 12(22): e4552-e4552.