Monitoring B Cell Activation with Antigenic Liposomes using a Calcium-Flux Assay
Published: November 30, 2023
Abstract
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.
Protocol
1. Conjugation of Protein Antigen to PEGylated Lipid 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. In a 1.0 cm x 30 cm glass chromatography column with the turnover stopper closed, add PBS to the column to was…
Representative Results
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 <em…
開示
The authors have nothing to disclose.
Materials
| 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 |
