This work was supported by NIH grant R15ES030140 and NSF grant CBET1903568. Financial support from the Russ College of Engineering and Technology and the Department of Chemical and Biomolecular Engineering at Ohio University is also acknowledged.

All human blood samples used in the protocol described below were purchased as de-identified samples. No human subjects were directly involved or recruited for this study. The guidelines of the Declaration of Helsinki should be used when research involves human subjects.
1. Erythrocyte isolation from whole blood
<ol>
	<li>Add 500 &#181;L of whole blood in acid citrate dextrose (ACD) (stored at 4 &#176;C) to a microcentrifuge tube. 
	NOTE: Whole blood was purchased in ACD. According to the company, 1.5 mL of ACD is added to 7 mL of whole blood (8.5 mL total volume).</li>
	<li>Centrifuge the whole blood at 700 x g for 5 min at room temperature (RT) and remove the clear plasma and the thin buffy coat using a pipette to leave the red erythrocyte layer.</li>
	<li>Prepare 1 L of Ringer solution containing 125 mM NaCl, 5 mM KCl, 1 mM MgSO4, 32 mM HEPES, 5 mM glucose, and 1 mM CaCl2. Adjust the pH to 7.4 by adding 2 &#181;L drops of 1.0 M NaOH. To prepare glucose-free Ringer solution, follow the same protocol, but do not include glucose in the solution.</li>
	<li>Wash the erythrocytes 2x in Ringer solution by suspending the cell pellet in 1.5 mL of Ringer solution, centrifuging at 700 x g for 5 min at RT, and removing the supernatant.</li>
	<li>Make a 0.4% hematocrit by resuspending 40 &#181;L of the erythrocyte pellet in 9,960 &#181;L of glucose-free Ringer solution to reach a final volume of 10 mL. 
	NOTE: Hematocrit is a term used to refer to the volume fraction of erythrocytes in suspension. A 0.4% hematocrit is a suspension containing 0.4% erythrocytes.</li>
	<li>Incubate the cell suspension at 37 &#176;C for 7 days.</li>
</ol>
2. Treatment of erythrocytes with ionomycin and measurement of hemolysis
<ol>
	<li>Dissolve 1 mg of ionomycin calcium salt in 630 &#181;L of dimethyl sulfoxide (DMSO) to reach a final concentration of 2 mM. Aliquot and store at -20 &#176;C.</li>
	<li>Take 1 mL of the 0.4% hematocrit from step 1.5 and add 0.5 &#181;L of 2 mM ionomycin to reach a final concentration of 1 &#181;M. Incubate for 2 h at 37 &#176;C.
	<ol>
		<li>Use 1 mL of the hematocrit with no ionomycin treatment as a negative control.</li>
	</ol>
	</li>
	<li>Centrifuge the ionomycin-treated and untreated hematocrits at 700 x g for 5 min at RT, and remove their supernatants to leave the cell pellets at the bottom of the tubes.</li>
	<li>Wash the cells 3x with Ringer solution by suspending the cell pellets in 1.5 mL of Ringer solution, centrifuging at 700 x g for 5 min at RT and discarding the supernatants.</li>
	<li>To measure hemolysis, add 1 mL of the untreated 0.4% hematocrit from step 1.5 to a microcentrifuge tube and incubate for 2 h at 37 &#176;C as the negative control for hemolysis (0%).</li>
	<li>Add 1 mL of the untreated 0.4% hematocrit from step 1.5 to a microcentrifuge tube and centrifuge at 700 x g for 5 min at RT. Remove the supernatant and add 1 mL of distilled water to the cell pellet and incubate for 2 h at 37 &#176;C as the positive control for hemolysis (100%).</li>
	<li>Add 1 mL of the ionomycin-treated 0.4% hematocrit from step 2.2 to a microcentrifuge tube.</li>
	<li>Centrifuge the untreated cells, treated cells, and the cells in distilled water at 700 x g for 5 min at RT.</li>
	<li>Take 200 &#181;L of the supernatants and add to a 96-well plate.</li>
	<li>Measure the absorbance at 541 nm using a microplate reader.</li>
	<li>Calculate the hemolysis using Equation 132: 
	 
	%Hemolysis = (AT - A0)/(A100 - A0)*100 
	 
	where A0 is the absorbance of erythrocytes in Ringer solution, A100 is the absorbance of erythrocytes in water, and AT is the absorbance of treated erythrocytes by ionomycin.</li>
</ol>
3. Annexin-V binding assay
<ol>
	<li>Dilute 2 mL of the 5x annexin V binding buffer in 8 mL of phosphate-buffered saline (PBS) to obtain 1x binding buffer.</li>
	<li>Resuspend the ionomycin-treated and untreated cell pellets from step 2.4 in 1 mL of 1x binding buffer.</li>
	<li>Take 235 &#181;L of the cell suspensions in the binding buffer and add 15 &#181;L of Annexin V-Alexa Flour 488 conjugate.</li>
	<li>Incubate the cells at RT for 20 min in a dark place. Centrifuge at 700 x g for 5 min at RT and remove the supernatant.</li>
	<li>Wash the cells 2x with 1x binding buffer, by suspending the cell pellet in 1.5 mL of the binding buffer, centrifuging at 700 x g for 5 min at RT and removing the supernatant.</li>
	<li>Resuspend the cell pellets in 250 &#181;L of 1x binding buffer for flow cytometry measurements.</li>
</ol>
4. Flow cytometry
<ol>
	<li>Transfer 200 &#181;L of the annexin-V stained erythrocytes to 1 mL round bottom polystyrene tubes compatible with flow cytometry.</li>
	<li>Login to the flow cytometry software and click on the new experiment button. Click on the new tube button. Select the global sheet and choose the apply analysis to measure the fluorescence intensity with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.</li>
	<li>Set number of cells to 20,000 to be collected for fluorescence-activated cell sorting (FACS) analysis.</li>
	<li>Select the desired tube and click on load button. Click on record button for forward scatter and side scatter measurements. Repeat for all samples.</li>
	<li>Right click on specimen button and click on apply batch analysis to generate the result file.</li>
	<li>Right click on specimen button and click on generate FSC files.</li>
	<li>Add the flow cytometry data (FSC files) into the workplace of flow cytometry software.</li>
	<li>Analyze the control data by selecting the cell population of interest and adding statistics for eryptosis value.
	<ol>
		<li>Double click on control and select histogram versus fluorescence intensity.</li>
		<li>Click on gate button to draw a gate on the histogram which indicates the percentage of eryptosis.</li>
	</ol>
	</li>
	<li>Apply the same statistics for all other experimental tubes to obtain the eryptosis values. Right click on control and select copy analysis to group.</li>
	<li>After properly gating all samples, transfer the analyzed data by dragging and dropping them into the layout editor.
	<ol>
		<li>Overlay the analyzed data with control in layout editor.</li>
		<li>Set the desired histograms and intensities by changing the x and y axis of the overlaid graphs.</li>
		<li>Export image files by clicking on export button and save the graphs in desired location.</li>
	</ol>
	</li>
</ol>
5. Confocal microscopy
<ol>
	<li>Transfer 5 &#181;L of annexin-V-stained cells on a microscope slide and cover it with a cover slip. Keep in a dark place to prevent photobleaching.</li>
	<li>Use Argon laser of the confocal fluorescence microscope to observe the cells excited at 488 nm with desired magnifications. 
	NOTE: A confocal microscope is not necessarily needed and any microscope with fluorescence capabilities can be used to obtain fluorescence images that demonstrate annexin-V binding.</li>
	<li>Obtain fluorescence images of the control (non-treated cells) and treated cells. 
	NOTE: Non-treated cells are expected to show very weak fluorescence signals, whereas treated cells are expected to show bright green fluorescence on their membranes.</li>
</ol>

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The authors have nothing to disclose.

The goal of this procedure is to provide optimal values for ionophore concentration, treatment time, and extracellular glucose concentration, which are important factors in ensuring successful induction of eryptosis. A critical step in the protocol is the depletion of extracellular glucose, which, despite its importance, has not been sufficiently emphasized in the literature. The sugar content in normal Ringer solution (5 mM) has an inhibitory effect on eryptosis. Glucose depletion in the extracellular environment induce...

Programmed cell death in erythrocytes, also known as eryptosis, is common in many clinical conditions and hematological disorders. Eryptosis is associated with cell shrinkage and the loss of phospholipid asymmetry in the cell plasma membrane1,2. Loss of asymmetry results in the translocation of phosphatidylserine (PS), a lipid normally localized in the inner leaflet3,4, to the cell outer leaflet, which signals to macrophages to phagocytose and remove defective erythrocytes5,6,7,8. At the end of the normal life span of erythrocytes, removal of eryptotic cells by macrophages ensures the balance of erythrocytes in circulation. However, in diseased conditions, such as sickle cell disease and thalassemia9,10,11, enhanced eryptosis may result in severe anemia2. Due to its importance in hematological diseases, there is significant interest in examining the factors inducing or inhibiting eryptosis and the molecular mechanisms underlying this process.
The plasma membrane of healthy erythrocytes is asymmetric, with different phospholipids localizing at the outer and inner leaflets. Membrane asymmetry is primarily regulated by the action of membrane enzymes. Aminophospholipid translocase facilitates the transport of aminophospholipids, PS and phosphatidylethanolamine (PE), by directing these lipids to the cell inner leaflet. On the other hand, floppase transports the choline containing phospholipids, phosphatidylcholine (PC) and sphingomyelin (SM), from the inner to the outer leaflet of the cell membrane12. However, unlike healthy cells, the membrane of eryptotic erythrocytes is scrambled. This is due to the action of a third enzyme, scramblase, which disrupts phospholipid asymmetry by facilitating the bidirectional transport of aminophospholipids13,14,15,16. Scramblase is activated by elevated intracellular levels of Ca2+. Therefore, calcium ionophores, which facilitate the transport of Ca2+ across the cell membrane12, are efficient inducers of eryptosis.
Ionomycin, a calcium ionophore, has been widely used to induce eryptosis in erythrocytes12,17,18,19,20,21,22,23,24,25,26. Ionomycin has both hydrophilic and hydrophobic groups, which are necessary to bind and capture Ca2+ ion, and transport it to the cytosolic space27,28,29. This leads to the activation of scramblase and translocation of PS to the outer leaflet, which can be easily detected using annexin-V, a cellular protein with a high affinity to PS12. Although triggering eryptosis by ionomycin is commonly reported, there is considerable method discrepancy in the literature (Table 1). The population of erythrocytes undergoing eryptosis depends on different factors such as ionophore concentration, treatment time with ionophore, and the sugar content of extracellular environment (glucose depletion activates cation channels and facilitates the entry of Ca2+ into the cytosolic space)30,31. However, there is little consistency in these factors in the literature, making it difficult to perform eryptosis reproducibly in vitro.
In this protocol, we present a step-by-step procedure to induce eryptosis in human erythrocytes. Factors affecting successful eryptosis including Ca2+ concentration, ionophore concentration, treatment time, and pre-incubation in glucose-depleted buffer are examined and optimal values are reported. This procedure demonstrates that pre-incubation of erythrocytes in a glucose-free buffer significantly increases the percentage of eryptosis compared to glucose-containing buffer. This protocol can be used in the laboratory to produce eryptotic erythrocytes for various applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plate</td>
			<td>Fisher Scientific</td>
			<td>12-565-331</td>
			<td></td>
		</tr>
		<tr>
			<td>Annexin V Alexa Fluor 488 - apoptosis kit</td>
			<td>Fisher Scientific</td>
			<td>A10788</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>BD FACSAria II flow cytometer</td>
			<td>BD Biosciences</td>
			<td>643177</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Millipore Sigma</td>
			<td>M7157</td>
			<td>Model Eppendorf 5415C</td>
		</tr>
		<tr>
			<td>Confocal fluorescence microscopy</td>
			<td>Zeiss, LSM Tek Thornwood</td>
			<td></td>
			<td>Model LSM 510, Argon laser excited at 488 nm for taking images</td>
		</tr>
		<tr>
			<td>Cover glasses circles</td>
			<td>Fisher Scientific</td>
			<td>12-545-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable round bottom flow cytometry tube</td>
			<td>VWR</td>
			<td>VWRU47729-566</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>472301-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>VWR Life Science</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose monohydrate</td>
			<td>Sigma-Aldrich</td>
			<td>Y0001745</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer (1 M)</td>
			<td>Fisher Scientific</td>
			<td>50-751-7290</td>
			<td>Store at 4 &#176;C</td>
		</tr>
		<tr>
			<td>Ionomycin calcium salt</td>
			<td>EMD Milipore Corp.</td>
			<td>407952-1MG</td>
			<td>Dissolve in DMSO to reach 2 mM. Store at -20 &#176;C</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fisher Scientific</td>
			<td>P330-500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Fisher Scientific</td>
			<td>M65-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Fisher Scientific</td>
			<td>02-681-5</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Plain glass microscope slides</td>
			<td>Fisher Scientific</td>
			<td>12-544-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HFM microplate reader</td>
			<td>BioTek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whole blood in ACD</td>
			<td>Zen-Bio</td>
			<td></td>
			<td>Store at 4 &#176;C and warm to 37 &#176;C prior to use</td>
		</tr>
	</tbody>
</table>

induction of eryptosis in red blood cells using a calcium ionophore

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic cells is the loss of compositional asymmetry of the cell membrane, leading to the translocation of phosphatidylserine to the membrane outer leaflet. This process is triggered by increased intracellular concentration of Ca2+, which activates scramblase, an enzyme that facilitates bidirectional movement of phospholipids between membrane leaflets. Given the importance of eryptosis in various diseased conditions, there have been efforts to induce eryptosis in vitro. Such efforts have generally relied on the calcium ionophore, ionomycin, to enhance intracellular Ca2+ concentration and induce eryptosis. However, many discrepancies have been reported in the literature regarding the procedure for inducing eryptosis using ionomycin. Herein, we report a step-by-step protocol for ionomycin-induced eryptosis in human erythrocytes. We focus on important steps in the procedure including the ionophore concentration, incubation time, and glucose depletion, and provide representative result. This protocol can be used to reproducibly induce eryptosis in the laboratory.

Optimization of ionomycin concentration
While ionomycin is required to induce eryptosis, increased ionomycin concentrations can lead to hemolysis (i.e. lysis of erythrocytes and release of hemoglobin), which needs to be avoided. Treatment of erythrocytes with 1 &#181;M ionomycin in Ringer solution for 2 h is enough to induce eryptosis, as evidenced by successful labeling with annexin-V Alexa Flour 488 conjugate ...

Watch this Scientific Journal Video about Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore at JoVE.com

Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided.

Eryptosis, erythrocyte programmed cell death, occurs in a number of hematological diseases and during injury to erythrocytes. A hallmark of eryptotic ...

induction-of-eryptosis-in-red-blood-cells-using-a-calcium-ionophore

Research

JoVE Journal

Biochemistry

8.6K Views.  Ohio University. A protocol for the induction of eryptosis, programmed cell death in erythrocytes, using the calcium ionophore, ionomycin, is provided. Successful eryptosis is evaluated by monitoring the localization phosphatidylserine in the membrane outer leaflet. Factors affecting the success of the protocol have been examined and optimal conditions provided.

Video: Induction of Eryptosis in Red Blood Cells Using a Calcium Ionophore

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for the targeted expression of genetically encoded calcium sensors. This protocol outlines the use of these sensors for the in vivo imaging of calcium dynamics in targeted cells, specifically the body wall muscles of the worms. By utilizing the co-expression of presynaptic channelrhodopsin, stimulation of acetylcholine release from excitatory motor neurons can be induced using blue light pulses, resulting in muscle depolarization and reproducible changes in cytoplasmic calcium levels. Two worm immobilization techniques are discussed with varying levels of difficulty. Comparison of these techniques demonstrates that both approaches preserve the physiology of the neuromuscular junction and allow for the reproducible quantification of calcium transients. By pairing optogenetics and functional calcium imaging, changes in postsynaptic calcium handling and homeostasis can be evaluated in a variety of mutant backgrounds. Data presented validates both immobilization techniques and specifically examines the roles of the C. elegans sarco(endo)plasmic reticular calcium ATPase and the calcium-activated BK potassium channel in the body wall muscle calcium regulation.

In Vivo Calcium Imaging in C. elegans Body Wall Muscles

This method provides a way to couple optogenetics and genetically encoded calcium sensors to image baseline cytosolic calcium levels and changes in evoked calcium transients in the body wall muscles of the model organism C. elegans.

The model organism C. elegans provides an excellent system to perform in vivo calcium imaging. Its transparent body and genetic manipulability allow for ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

SUMO-Binding Entities (SUBEs) as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in liver cancer, the modification by SUMO (Small Ubiquitin MOdifier) has been given the most attention. Isolation of endogenous SUMOylated proteins in vivo is challenging due to the presence of active SUMO-specific proteases. Initial studies of SUMOylation in vivo were based on the molecular detection of specific SUMOylated proteins (e.g., by western blot). However, in many cases, antibodies, generally made with non-modified recombinant protein, did not immunoprecipitate SUMOylated forms of the protein of interest.&#160;Nickel chromatography has been the other approach to study SUMOylation by capturing histidine-tagged versions of SUMO molecules. This approach is mainly used in cells stably expressing or transiently transfected with His-SUMO molecules. To overcome these limitations, SUMO-binding entities (SUBEs) were developed to isolate endogenous SUMOylated proteins. Herein, we describe all the steps required for the enrichment, isolation, and identification of SUMOylated substrates from human hepatoma cells and hepatic tissues from a liver cancer mouse model by using SUBEs. Firstly, we describe methods involved in the preparation and lysis of the human hepatoma cells and liver tumor tissue samples. Then, a thorough explanation of the preparation of SUBEs and controls is detailed along with the protocol for the protein pull-down assays. Finally, some examples are provided regarding the options available for the identification and characterization of the SUMOylated proteome, namely the use of western-blot analysis for the detection of a specific SUMOylated substrate from liver tumors or the use of proteomics by mass spectrometry for high-throughput characterization of the SUMOylated proteome and interactome in hepatoma cells.

SUMO-Binding Entities SUBEs as Tools for the Enrichment, Isolation, Identification, and Characterization of the SUMO Proteome in Liver Cancer

Here, we present a protocol to enrich, isolate, identify, and characterize proteins modified by SUMO in vivo both from human hepatoma cells and liver tumors obtained from mouse models of hepatocellular carcinoma by using SUMO-binding entities (SUBEs).

Post-translational modification is a key mechanism regulating protein homeostasis and function in eukaryotic cells. Among all ubiquitin-like proteins in ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

Immunostaining-Based Detection of Dynamic Alterations in Red Blood Cell Proteins

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute alterations in protein activation states. It facilitates the assessment of RBC treatments, characterization of differences in certain disease states, and description of cellular coherencies. The detection of acutely altered protein activation (e.g., through mechanotransduction) requires adequate sample preparation to preserve otherwise temporary protein modifications. The basic principle includes immobilizing the target binding sites of the desired RBC proteins to enable the initial binding of specific primary antibodies. The sample is further processed to guarantee optimal conditions for the binding of the secondary antibody to the corresponding primary antibody. The selection of non-fluorescent secondary antibodies requires additional treatment, including biotin-avidin coupling and the application of 3,3-diaminobenzidine-tetrahydrochloride (DAB) to develop the staining, which needs to be controlled in real-time under a microscope in order to stop the oxidation, and thus staining intensity, on time. For staining intensity detection, images are taken using a standard light microscope. In a modification of this protocol, a fluorescein-conjugated secondary antibody can be applied instead, which has the advantage that no further development step is necessary. This procedure, however, requires a fluorescence objective attached to a microscope for staining detection. Given the semi-quantitative nature of these methods, it is imperative to provide several control stains to account for non-specific antibody reactions and background signals. Here, we present both staining protocols and the corresponding analytical processes to compare and discuss the respective results and advantages of the different staining techniques.

Capturing dynamic changes in the protein activation of enucleated red blood cells poses methodological challenges, like the preservation of dynamic changes to acute stimuli for later assessment. The presented protocol describes sample preparation and staining techniques that enable preservation and analysis of relevant protein changes and subsequent detection.

Antibody labeling of red blood cell (RBC) proteins is a commonly used, semi-quantitative method to detect changes in overall protein content or acute ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...