Monitoring B Cell Activation with Antigenic Liposomes using a Calcium-Flux Assay

1. Conjugation of Protein Antigen to PEGylated Lipid

1. Add 3 g of cross-linked dextran gel beads with a fractionation range of 1,500–30,000 Daltons (Da) to a 250 mL vacuum flask. Add 60 mL of phosphate-buffered saline (PBS) and continually stir with a magnetic stir bar. Seal the flask and attach it to a vacuum to initiate de-gassing on a stir plate for a minimum of 1 h.
2. In a 1.0 cm x 30 cm glass chromatography column with the turnover stopper closed, add PBS to the column to wash. Open the turnover stopper and drain the column. Following washing, add the slurry of de-gassed beads to the column. Continually add the slurry until the column is packed to a bead height of approximately 24 cm.
3. Once the column has a 'head' of approximately 4–5 cm of PBS above the packed beads, have the following steps ready.
   1. Create a gravity flow siphon by measuring a piece of tubing long enough to reach a shelf 1–2 feet above the top of the column and create a loop that drops near the bottom of the column and returns to the top of the column. Add a turnover stopper to one end of the tubing, leaving one side of the tubing open. Place the open tube end into a 500 mL bottle of PBS and place it on the shelf above the column.
   2. Take the end with the turnover stopper and attach a 10 mL syringe. Open the turnover stopper valve on the tubing line and draw PBS through the tube. Once the PBS has reached three-quarters of the way through the tubing, quickly remove the syringe and add the turnover stopper to the top of the column — the liquid should be running onto the column.   
      NOTE: The amount of 'head' of PBS left on the top of the column should be between 1 and 3 cm. The minimal amount to wash the column is 3 column volumes.
4. Determine the amount of protein (moles) to be linked to lipids based on its molecular weight (MW).
   NOTE: The Fab fragment of goat anti-mouse immunoglobulin, IgM (itself an IgG) is used here as a universal surrogate antigen for polyclonal stimulation of B-cells. Accordingly, the Fab fragment of goat IgG has a MW of approximately 48 kiloDalton (kDa) and is commercially available in a total quantity of 1.3 mg. Thus, the amount of protein to be linked is 27.1 µmol.
5. Determine the extinction coefficient of the protein of interest. If unknown, measure the protein absorbance at 280 nm using a spectrophotometer and divide the A₂₈₀ value by the number of moles calculated in step 1.4.
   NOTE: The extinction coefficient of Fab of goat IgG at 280 nm is 64,600 M^-1.
6. To ensure that trace amounts of the amine-containing buffer are removed, desalt the protein on the column created in steps 1.1–1.3 and collect protein fractions into 1.5 mL microcentrifuge tubes (~500 µL per fraction).
   1. Close the turnover stopper valve connected to the top of the column and remove the top of the column. If not already open, open the turnover stopper valve at the bottom of the column and wait until the 'head' of PBS hits the top of the beads.
   2. Slowly add the protein of interest to the top of the beads using a glass Pasteur pipette, being careful to minimize disturbance to the top of the beads.
   3. Once the 'head' of the protein solution has reached the top of the beads, add 1 mL of PBS gently via a Pasteur pipette. Repeat three times. Add PBS to create a head of 4–5 cm and repeat step 1.3 to wash the column.
      NOTE: This step can be skipped if it is certain that no amine-containing buffer is present.
7. Determine the fractions that contain protein by measuring A280 values and pool the fractions that give approximately 90% recovery of the protein.        
   NOTE: This typically dilutes the protein 1.5-2-fold. Determine the protein concentration after pooling. Concentrate the protein at this step, if necessary, using a centrifugation concentrator.
8. Add 2.5 molar equivalent of the heterobifunctional crosslinker to the protein.
   1. First, weigh approximately 5 mg of succinimidyl 3-(2-pyridyldithio)propionate (SPDP, MW = 314 g/mol) into a microcentrifuge tube. To determine the volume to dissolve the SPDP, takmultiplye molar protein concentration and250x to generate a 100x solution that will give a 2.5 molar excess in the reaction.      
      NOTE: For example, if the protein concentration is 50 µM, a solution of SPDP at 12.5 mM is needed. For 5 mg of SPDP, this corresponds to 1.27 mL of dimethyl sulfoxide (DMSO).
9. Add SPDP to the protein of interest at a 1:100 dilution. Place the reaction on an oscillating shaker at room temperature (RT) for approximately 1 h.
10. To remove excess SPDP and protect the endogenous disulfide bond(s), in the protein in the subsequent reduction step, equilibrate, and wash the column created in steps 1.1–1.3 in 100 mM sodium acetate (NaOAc) at pH 5.5 or create an identical column solely for use in this buffer.
11. After 1 h incubation with SPDP, desalt the protein on the column equilibrated in 100 mM NaOAc. Carry out this step in an identical manner as step 1.6 except using 100 mM NaOAc (pH 5.5) as the eluent. Collect the fractions, determine the A₂₈₀, and pool the top fractions. Wash the column in PBS.
12. Prepare a solution of 2.5 M of DTT (MW = 154 g/mol) in double distilled water (ddH₂O). Add 25 mM DTT to the pooled fractions of protein for 5-10 min.
13. Measure the A₂₈₀ of the protein and divide this by the extinction coefficient. Also, determine the A₃₄₃. Calculate the linking ratio based on the molarity of protein (A₂₈₀) and linker (A₃₄₃).        
    NOTE: Turn off the A340 auto-calibration if using a nanodrop spectrophotometer. The A₃₄₃ measurement represents the absorbance of the pyridine 2-thione group (extinction coefficient = 7550 M^-1) that is removed from the SPDP cross-linker by the DTT. A molar ratio of linker: protein ratio of 1.2–1.5 is generally achieved when performing the reaction with a 2.5 molar excess of SDPD on a wide range of proteins.
14. Run the pooled fractions over the column washed in PBS as described above. Collect the fractions, determine the A₂₈₀, and pool top fractions. Determine the concentration of protein by A280 after pooling the fractions.
15. Prepare DSPE-PEG(2000)-Maleimide (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000)-Maleimide. This will be added to the protein at a 10:1 molar ratio. Prepare a 100x stock of DSPE-PEG(2000)-Maleimide to achieve the desired concentration.
    1. For example, if the protein concentration is 10 µM after step 1.14, prepare a stock of DSPE-PEG(2000)-Maleimide at 10 mM. Carefully weigh out DSPE-PEG(2000)-Maleimide (MW = 3000 g/mol) and add the appropriate amount of DMSO. Multiple rounds of gentle vortexing and sonicating may get it fully dissolved.
16. Determine the total volume of the fractions from step 1.14 and transfer the solution carefully into a small (10-25 mL) round bottom flask (RBF). Add the appropriate amount of 100x DSPE-PEG(2000)-Maleimide to the protein to achieve a 1x, 10-fold molar excess, final concentration. Gently swirl the solution to ensure that the DMSO is fully dispersed.
17. Run reaction overnight under nitrogen in a sealed RBF. Use a rubber septum and place it in a fume hood.
    1. Remove the atmosphere by puncturing the septum with a 20 G needle attached to a 3 mL syringe at the end of the tubing attached to a vacuum, and turn on the vacuum for 3–5 s.
    2. Replace the atmosphere with nitrogen. Fill a balloon attached to a 3 mL syringe cut in half with nitrogen and attach a 20 G needle. Puncture the septum to fill the atmosphere inside the round bottom flask with nitrogen. Repeat the removal of the atmosphere and replacement with nitrogen once more and leave the nitrogen balloon attached overnight.
18. Prepare a separate cross-linked dextran gel bead column with a fractionation range of 4,000–150,000 Da in a 1.0 cm x 50 cm glass chromatography column (as per steps 1.1-1.3).
19. The following day, remove the protein from the round bottom flask prepared in step 1.17 and run (follow the method used in step 1.6) over the cross-linked dextran gel bead column (from step 1.18).   
    NOTE: The total amount to load onto the column should be no more than 2 mL. If a larger reaction was used, split into two and run on separate columns or run on a larger diameter column.
    1. Collect the fractions, determine the A₂₈₀, pool top fractions, and determine the concentration of protein. The presence of the lipid will not affect the measurements.
20. Store the final pooled fractions of lipid-linked protein at 4 °C until ready for use.

2. Liposome Preparation

1. Weigh out the lipids: DSPE-PEG(2000) (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000, MW = 2805.5 g/mol; Cholesterol (3b-Hydroxy-5-cholestene, 5-Cholesten-3b-ol), MW = 386.7 g/mol; and DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), MW = 790.1 g/mol. Create a 5 mg/mL solution of DSPC, 4 mg/mL solution of Cholesterol, and 2 mg/mL solution of DSPE-PEG(2000) in chloroform.
2. Ensure that the molar ratio of lipids in the liposomes is: 57% DSPC, 38% Cholesterol, and 5% DSPE-PEG(2000), as represented in Table 1.
   NOTE: It is important to control the amount of antigen on the liposome, which generally ranges from 0.01–0.1%. In Table 1, 0.1% of goat anti-mouse IgM Fab fragment linked to PEGylated lipid is used. It is critical to take into account the 10-fold molar excess of DSPE-PEG(2000)-Maleimide that was added to the protein in step 1.15, to ensure that the total amount of PEGylated lipid does not exceed 5%.
3. Consider the final lipid amounts (µmol) when creating lipids for extrusion. See Table 1 for an example of calculating molar lipid concentrations for the creation of liposomes at a 1.25 µM total lipid concentration.
4. Once the correct amount of each lipid to be added together has been calculated, combine all into a 12 mL borosilicate glass test tube.
5. Carefully blow off the chloroform.
   1. Use the tubing and a 3 mL syringe with a needle, prepared in step 1.17.1, to carefully blow off chloroform in the tube with nitrogen. Gently blow a stream of nitrogen on the solution while rotating the tube with the other hand. Be careful to minimize splashing or causing the liquid to run way up the side of the tube as it may be more difficult to get this into solution in the next step.
6. Add 100 µL of DMSO to each tube and lyophilize this solution overnight. Cover the top of the tube with a non-static laboratory wipe, secure it with a rubber band, and freeze at -80 °C.

3. Liposome Extrusion

1. Add 1 mL of PBS containing the lipid-linked protein to the 12 mL tube containing the lipids. Sonicate the solution for approximately 30 seconds to 1 minute in a sonication water bath. Rest for 5 min or longer between each round and repeat 3–4 times.
2. Prepare an extruder as directed by the manufacturer.
   1. Add the filter supports to the internal membrane support, and place a few drops (10 µL each) of PBS on paraffin film. Using tweezers, grab an edge of the filter support and dip it in the 10 µL drop.
   2. Place the filter inside the O-ring. Do this for both sides. Using tweezers, grab a 0.8 µm filter on the edge dip in the 10 µL drop, and coat the outside of the O-ring with PBS. Place the 0.8 µm filter gently on the O-ring so the filter does not overhang the internal membrane support.
3. Place the extruder heating block onto a hot plate to pre-heat the extruder. Switch the hot plate on low and allow the extruder heating block to reach the desired temperature. To avoid potential issues with protein aggregation, do not heat the block past 37 °C.
4. Load the sonicated sample into one of the extruding syringes placed into the extruder. Place the extruder syringe into the other end of the extruder. Make sure the empty syringe plunger is set to zero. The empty syringe will fill as the lipid is extruded through the polycarbonate 0.8 µm membrane. Try to avoid passing bubbles back and forth through the membrane.
5. Place the fully assembled extruder into the heating block. Gently push the plunger of the filled syringe with lipids to the empty syringe. Depending on the lipid concentration, this will be difficult to push through. Repeat 20x.
6. Remove the fully assembled extruder from the heating block. Slowly remove the filled syringe from the extruder, be sure to collect any lipids that may leak out when removing. Place liposomes into a clean vial. Repeat steps 3.4–3.6 using polycarbonate 0.2 µm and 0.1 µm membranes.
7. Create a 0.7 x 50 cm² cross-linked agarose bead column with a separation range of 0.7-10 Mega Daltons (MDa) equilibrated in PBS. Add the liposomes and run as in step 1.6. Fractions containing liposomes will have decreased transmission of light in the 250–400 nm range.
8. Store the liposomes at 4 °C and do not freeze.

4. Calcium Flux to Monitor B-cell Activation by Antigenic Liposomes

1. Euthanize mice by carbon dioxide (CO₂) or isoflurane overdose, according to institutional standard operating procedures. Confirm euthanasia of mice by cervical dislocation.
2. Sterilize surgical instruments and the mice carcass using 70% ethanol. Use 10 cm long, 0.8 mm tip, curved iris forceps to separate the skin covering the chest cavity from the rib cage. Use 10 cm long, straight dissecting scissors to make a distal incision with a bilateral incision.
3. Use curved iris forceps and dissecting scissors to extract the spleen from the abdominal cavity. Remove fatty tissue surrounding the spleen and then place it in a 15 mL polystyrene conical tube filled with RPMI1640 medium containing 1% fetal calf serum (FCS), and penicillin-streptomycin.
4. Transfer spleen and media over a 40 µm cell strainer fitted on a 50 mL conical tube. Using the rubber end of a 3 mL syringe plunger, gently crush the spleen. Wash the 40 µm cell strainer with the media. Repeat this process until only fat tissue is left in the 40 µm cell strainer. Rinse the sieve with 3-5 mL of media to increase cell recovery.
5. Centrifuge cell homogenate at 300 x g for 5 min at RT. After centrifugation, discard the supernatant and add 10 mL of 1x RBC lysis buffer to the pellet. Pipette up and down, leave for 2–3 min at RT, and then centrifuge at 300 x g for 5 min at RT.
6. Discard the supernatant and resuspend red blood cell-depleted splenocytes in 10 mL of media. Determine total cell numbers using a hemocytometer or other cell counting device. Pellet the cells by centrifugation as described above.
   NOTE: The ideal final concentration necessary for Indo-1 loading of splenocytes is 10–20 x 10⁶ cells/mL, but a lower concentration of cells can be used.
7. Resuspend splenocytes at 15 x 10⁶ cells/mL in calcium flux loading buffer (Roswell Park Memorial Institute, RPMI1640 medium containing 1% FCS, 10mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1mM magnesium chloride (MgCl₂), 1mM ethylene glycol-bis(β-aminoethyl ether)-N, N,N', N'-tetraacetic acid (EGTA), and 5% penicillin-streptomycin). Add in 1.5 µM Indo-1 from a 1 mM DMSO stock solution, and invert the tube several times to mix. Protect from light, and incubate cells at 37 °C water bath for 30 min.
8. Following the 30-minute incubation, add in 5x the amount of calcium flux loading buffer. Centrifuge at 300 x g for 7 min at RT.
9. For gating on B cells, stain cells with 1:200 anti-mouse CD5-PE and anti-mouse 1:200 B220-PE/Cy7 in 0.5 mL of loading buffer at 4 °C for 20 min, protected from light.
10. Wash splenocytes in calcium flux loading buffer and centrifuge at 300 x g for 7 min at RT. Resuspend cells at 10–20 x 10⁶ cells/mL in calcium flux running buffer (Hank's balanced salt solution (HBSS) containing 1% FCS, 1 mM MgCl₂ , and 1 mM calcium chloride (CaCl₂)). Store on ice, protected from light until ready to run on the flow cytometer.
11. Set up flow cytometry with unstained and single stain controls for compensation.
    1. Run the stained cells to set up the gates appropriately (see Figure 1A). Setup Indo-1 (violet) versus Indo-1 (blue) plot, adjust the voltages in the Indo-1 channels to place the cells staining at a slope of 45–60° to maximize the signal: noise ratio of the Indo-1 violet: blue change in ratio following B-cell stimulation.
       NOTE: Make sure that the voltages are not too high, such that a significant percentage of the cells (>5%) are up against the top of the plot.
    2. Create a diagonal gate that encapsulates the top left portion (Violet⁺Blue^–) of the plot, such that without stimulation <10% of the cells are in the gate, which should ideally increase to >75% following stimulation.
12. Add 0.5 mL of cells to a capped 5 mL round bottom polystyrene tube (fluorescence-activated cell sorting (FACS) tube) and warm the cells to 37 °C for 3–5 min in a water bath. Place the FACS tube in the 37 °C water-jacketed chamber that is connected to a re-circulating water bath.
    1. Run the tube in the water jacket on the flow cytometer and initiate acquisition, without storing the data, at approximately 5,000–10,000 events/s. Once the cells have stabilized (15–30 s), initiate data acquisition and collect data for at least 10 s to establish the background.
    2. At the 10 snot mark, quickly remove the tube from the flow cytometer, add the stimulation (5-50 μM antigenic liposomes in 2–20 μL), pulse vortex, and return the FACS tube onto the flow cytometer. Maintain data acquisition and storage through this time and collect data for 3–5 min.
13. Analyze data in appropriate analysis software under the kinetics functions.

Table 1: Calculations for creating a 1.25 µM total lipid concentration liposome. Example of calculation table for goat anti-mouse IgM F(ab) fragment liposomes.