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

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

  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 was.......

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Figure 1
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 

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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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