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In This Article

  • Overview
  • Protocol
  • Results
  • Disclosures
  • Materials

Overview

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

  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 unti....

Results

figure-results-58
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 

Disclosures

No conflicts of interest declared.

Materials

NameCompanyCatalog NumberComments
Model 2110 Fraction CollectorBioRad7318122
CholestrolSigmaC8667Sigma grade 99%
SPDPThermo Fisher Scientific21857
DSPCAvanti850365
DSPE-PEG 18:0Avanti880120
DSPE-PEG MaleimideAvanti880126
ExtruderAvanti6100001mL syringe with holder/heating block
Filters 0.1 µmAvanti610005
Filters 0.8 µmAvanti610009
10mm Filter SupportsAvanti6100014
Glass Round Bottom FlaskSigmaZ100633
Turnover stoppersThermo Fisher ScientificP-301398
TubingThermo Fisher ScientificP-198194
Leur LockThermo Fisher Scientifick4201634503
Sephadex G50 BeadsGE Life Sciences17004201
Sephadex G100 BeadsGE Life Sciences17006001
Heat Inactivated Fetal Calf SerumThermo Fisher Scientific10082147
HEPES (1M)Thermo Fisher Scientific15630080
EGTASigmaE3889
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher Scientific15140122
1x RBC lysis BufferThermo Fisher Scientific00-4333-57
Indo-1InvitrogenI1203
CD5-PEBioLegend100608
B220-PE-Cy7BioLegend103222
HBSSThermo Fisher Scientific14170112Without calcium and magnesium
MgCl2SigmaM8266
CaCl2SigmaC4901
Fab anti-mouse IgMJackson ImmunoResearch115-007-020
F(ab')2 anti-mouse IgMJackson ImmunoResearch115-006-020

This article has been published

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Source: Bednar, K. J. et al., Antigenic Liposomes for Generation of Disease-specific Antibodies. J. Vis. Exp. (2018)

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