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

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

To monitor B cell response to antigenic liposome activation, resuspend 1.5 x 107 splenocytes in calcium flux loading buffer, and add 1.5 micromolar Indo-1 to the cells, inverting the solution in the tube several times to mix. After a 30-minute water bath incubation at 37 degrees Celsius protected from light, add 5 times the volume of calcium flux loading buffer, and centrifuge the Indo-1-labeled cells.
For B cell gating, stain cells with anti-CD5 and anti-B220 antibodies in 0.5 milliliters of loading buffer at 4 degrees Celsius for 20 minutes protected from light. At the end of the incubation, wash the cells in fresh calcium flux loading buffer, and resuspend the pellet at 1 to 2 x 107 cells per milliliter of fresh calcium flux running buffer for storage on ice protected from light, until analysis.
Next, add 0.5 milliliters of cells to a capped 5-milliliter round-bottom polystyrene tube, and warm the cells to 37 degrees Celsius in a water bath. After 3 to 5 minutes, transfer the tube to a 37 degrees Celsius water-jacketed chamber connected to a recirculating water bath, and run the tube in the water jacket on the flow cytometer.
After collecting 5,000 to 10,000 events per second, allow the cells to stabilize for 15 to 30 seconds, and re-initiate the data acquisition, collecting the data for at least 10 seconds to establish a background reading. At the 10-second mark, quickly remove the tube from the flow cytometer, and add the appropriate experimental concentration of antigenic liposomes. Then, pulse vortex the cells, and read the tube on the cytometer for an additional 3 to 5 minutes.

monitoring-b-cell-activation-with-antigenic-liposomes-using-calcium

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

Specialized Assays

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	<tbody>
		<tr>
			<td>Model 2110 Fraction Collector</td>
			<td>BioRad</td>
			<td>7318122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cholestrol</td>
			<td>Sigma</td>
			<td>C8667</td>
			<td>Sigma grade 99%</td>
		</tr>
		<tr>
			<td>SPDP</td>
			<td>Thermo Fisher Scientific</td>
			<td>21857</td>
			<td></td>
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		<tr>
			<td>DSPC</td>
			<td>Avanti</td>
			<td>850365</td>
			<td></td>
		</tr>
		<tr>
			<td>DSPE-PEG 18:0</td>
			<td>Avanti</td>
			<td>880120</td>
			<td></td>
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		<tr>
			<td>DSPE-PEG Maleimide</td>
			<td>Avanti</td>
			<td>880126</td>
			<td></td>
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			<td>Extruder</td>
			<td>Avanti</td>
			<td>610000</td>
			<td>1mL syringe with holder/heating block</td>
		</tr>
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			<td>Filters 0.1 &#181;m</td>
			<td>Avanti</td>
			<td>610005</td>
			<td></td>
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		<tr>
			<td>Filters 0.8 &#181;m</td>
			<td>Avanti</td>
			<td>610009</td>
			<td></td>
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			<td>10mm Filter Supports</td>
			<td>Avanti</td>
			<td>6100014</td>
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			<td>Glass Round Bottom Flask</td>
			<td>Sigma</td>
			<td>Z100633</td>
			<td></td>
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			<td>Turnover stoppers</td>
			<td>Thermo Fisher Scientific</td>
			<td>P-301398</td>
			<td></td>
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			<td>Tubing</td>
			<td>Thermo Fisher Scientific</td>
			<td>P-198194</td>
			<td></td>
		</tr>
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			<td>Leur Lock</td>
			<td>Thermo Fisher Scientific</td>
			<td>k4201634503</td>
			<td></td>
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			<td>Sephadex G50 Beads</td>
			<td>GE Life Sciences</td>
			<td>17004201</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G100 Beads</td>
			<td>GE Life Sciences</td>
			<td>17006001</td>
			<td></td>
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			<td>Heat Inactivated Fetal Calf Serum</td>
			<td>Thermo Fisher Scientific</td>
			<td>10082147</td>
			<td></td>
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			<td>HEPES (1M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
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			<td>EGTA</td>
			<td>Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>1x RBC lysis Buffer</td>
			<td>Thermo Fisher Scientific</td>
			<td>00-4333-57</td>
			<td></td>
		</tr>
		<tr>
			<td>Indo-1</td>
			<td>Invitrogen</td>
			<td>I1203</td>
			<td></td>
		</tr>
		<tr>
			<td>CD5-PE</td>
			<td>BioLegend</td>
			<td>100608</td>
			<td></td>
		</tr>
		<tr>
			<td>B220-PE-Cy7</td>
			<td>BioLegend</td>
			<td>103222</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Thermo Fisher Scientific</td>
			<td>14170112</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr>
			<td>Fab anti-mouse IgM</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-007-020</td>
			<td></td>
		</tr>
		<tr>
			<td>F(ab&#39;)2&#160;anti-mouse IgM</td>
			<td>Jackson ImmunoResearch</td>
			<td>115-006-020</td>
			<td></td>
		</tr>
	</tbody>
</table>

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...

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.

32 Aufrufe. Überwachung der B-Zell-Aktivierung mit antigenen Liposomen mit einem Calcium-Flux-Assay

Serie: Überwachung der B-Zell-Aktivierung mit antigenen Liposomen mit einem Calcium-Flux-Assay - Experiment

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Identifizierung von Inositolphosphat oder Phosphoinositid-Wechselwirkungsproteinen durch Affinitätschromatographie gekoppelt an Western Blot oder Massenspektrometrie

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

Forschung

JoVE Journal

Biochemie

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

Zellfreie Produktion von Proteoliposomen für die funktionelle Analyse und Antikörperentwicklung gegen Membranproteine

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Targeting Drogen zu Larval Zebrafish Makrophagen durch Injektion von Drogen-geladenen Liposomen

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

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

Transkript

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