Source: Bednar, K. J. et al., Antigenic Liposomes for Generation of Disease-specific Antibodies. J. Vis. Exp. (2018)
This video showcases the assessment of B cell activation through the utilization of antigenic liposomes. B cells treated with calcium-sensitive Indo-1 dye are distinguished using flow cytometry, and their violet-to-blue fluorescence ratio is analyzed. As the cells encounter antigenic liposomes, it triggers B cell activation through calcium influx, progressively enhancing the violet-to-blue fluorescence signal ratio over time.
1. Conjugation of Protein Antigen to PEGylated Lipid
2. Liposome Preparation
3. Liposome Extrusion
4. Calcium Flux to Monitor B-cell Activation by Antigenic Liposomes
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.
MW | 790 | 387 | 2900 | 3000 | 48000 | |
DSPC | Cholesterol | PEG-DSPE | excess PEG-DSPE | aIgM-PEG-DSPE | TOTAL | |
Molar ratio | 57 | 38 | 3.9 | 1 | 0.1 | 100.00 |
mass (mg) | 0.56 | 0.18 | 0.14 | 0.04 | 0.06 | 0.98 |
mmol | 0.71 | 0.47 | 0.05 | 0.01 | 0.00 | 1.25 |
mL | 112.50 | 45.93 | 70.64 | 0.00 | 525.97 | 755.03 |
Conc. (mg/mL) | ||||||
DSPC | 5 | |||||
Cholesterol | 4 | |||||
PEG-DSPE | 2 | |||||
aIgM-PEG-DSPE | 0.114 |
Figure 1: Representative gating strategy, calcium flux results, and analysis. (A) Cells were analyzed through the following gating strategy: live lymphocytes (FSC-A vs. SSC-A), single cells (FSC-W vs. SSC-W), and B-cells (B220+CD5–). (B) The Indo-1/Ca2+ flux response (violet vs. blue) of the B-cells. Shown is the calcium flux for anti-IgM Fab fragment liposomes and anti-IgM F(ab')2 at the same protein concentration (2.5 µg/mL) as well as buffer-stimulated cells as a control. Note that between 10-22 s is when the stimulation is added and, therefore, no data is acquired during this time.
The authors have nothing to disclose.
Model 2110 Fraction Collector | BioRad | 7318122 | |
Cholestrol | Sigma | C8667 | Sigma grade 99% |
SPDP | Thermo Fisher Scientific | 21857 | |
DSPC | Avanti | 850365 | |
DSPE-PEG 18:0 | Avanti | 880120 | |
DSPE-PEG Maleimide | Avanti | 880126 | |
Extruder | Avanti | 610000 | 1mL syringe with holder/heating block |
Filters 0.1 µm | Avanti | 610005 | |
Filters 0.8 µm | Avanti | 610009 | |
10mm Filter Supports | Avanti | 6100014 | |
Glass Round Bottom Flask | Sigma | Z100633 | |
Turnover stoppers | Thermo Fisher Scientific | P-301398 | |
Tubing | Thermo Fisher Scientific | P-198194 | |
Leur Lock | Thermo Fisher Scientific | k4201634503 | |
Sephadex G50 Beads | GE Life Sciences | 17004201 | |
Sephadex G100 Beads | GE Life Sciences | 17006001 | |
Heat Inactivated Fetal Calf Serum | Thermo Fisher Scientific | 10082147 | |
HEPES (1M) | Thermo Fisher Scientific | 15630080 | |
EGTA | Sigma | E3889 | |
Penicillin-Streptomycin (10,000 U/mL) | Thermo Fisher Scientific | 15140122 | |
1x RBC lysis Buffer | Thermo Fisher Scientific | 00-4333-57 | |
Indo-1 | Invitrogen | I1203 | |
CD5-PE | BioLegend | 100608 | |
B220-PE-Cy7 | BioLegend | 103222 | |
HBSS | Thermo Fisher Scientific | 14170112 | Without calcium and magnesium |
MgCl2 | Sigma | M8266 | |
CaCl2 | Sigma | C4901 | |
Fab anti-mouse IgM | Jackson ImmunoResearch | 115-007-020 | |
F(ab')2 anti-mouse IgM | Jackson ImmunoResearch | 115-006-020 |