eoe sub articles

EoE Sub Articles

1. Conjugation of Protein Antigen to PEGylated Lipid
<ol>
	<li>Add 3 g of cross-linked dextran gel beads with a fractionation range of 1,500&#8211;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.</li>
	<li>In a 1.0 cm x 30 cm glass chromatography column with the turnover stopper closed, add PBS to the column to was...

monitoring b cell activation with antigenic liposomes using a calcium-flux assay

Source: Bednar, K. J.&#160;et al.,&#160;<a href="https://app.jove.com/v/58285">Antigenic Liposomes for Generation of Disease-specific Antibodies.</a>&#160;J. Vis. Exp.&#160;(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.

<img alt="Figure 1" src="/files/ftp_upload/21873/21873_Figure1.jpg" /> 
Figure 1: Representative gating strategy, calcium flux results, and analysis.&#160;(A) Cells were analyzed through the following gating strategy: live lymphocytes (FSC-A&#160;vs.&#160;SSC-A), single cells (FSC-W&#160;vs.&#160;SSC-W), and B-cells (B220+CD5-). (B) The Indo-1/Ca2+&#160;flux response (violet&#160;<em...

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

1. Conjugation of Protein Antigen to PEGylated Lipid

	Add 3 g of cross-linked dextran gel beads with a fractionation range of 1,500&#8211;30,000 Daltons ...

Take red blood cell-depleted splenocytes in a buffer containing a cell-permeant calcium -sensitive fluorescent dye, Indo-1 AM.
Intracellular esterases cleave Indo-1 AM into membrane-impermeable Indo-1, which binds to calcium ions, changing Indo-1&#39;s fluorescence signal from blue to violet.
Introduce fluorophore-conjugated antigen-specific antibodies that bind specifically to the respective antigens on cells.
Using flow cytometry, identify fluorophore-labeled B cells and measure their Indo-1 violet-to-blue fluorescence ratio.
Now, treat the cells with antigenic liposomes comprising IgM Fab fragments conjugated to liposome bilayers.
The B cell receptors bind to the liposome-coupled antigens, activating the associated cytoplasmic tyrosine kinases and triggering phospholipase-C gamma-2 activation.
Activated phospholipase-C cleaves the membrane phospholipid, phosphatidylinositol&#160; 4,5-bisphosphate, generating second messengers, including inositol&#160; 1,4,5- trisphosphate, or IP3.
IP3 binds to IP3-gated calcium channels on the endoplasmic reticulum, releasing calcium ions into the cytoplasm.
These calcium ions bind the unbound Indo-1 molecules, increasing violet fluorescence.
A higher Indo-1 violet-to-blue fluorescence ratio in labeled B cells confirms their successful activation by antigenic liposomes.