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

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…

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