Finanziamenti dal NIH R01 HL56621.

Neutrofili isolamento Protocollo <ol><li> Portare i reagenti a temperatura ambiente. </li><li> Raccogliere 5,0 ml di terreni di isolamento dei neutrofili in provetta da centrifuga. Con attenzione strato 5,0 ml di sangue sui mezzi di separazione. Effettuare questo passaggio, lentamente e con attenzione, e con la punta della pipetta vicino alla superficie del supporto per evitare di mescolare il sangue e dei media. </li><li> Centrifugare a 500 RCF per 35 minuti a 20-25 ° C. Il sangue deve separare in 6 fasce distinte: al plasma, monociti, terreni di isolamento, i neutrofili, terreni di isolamento di più, e il rosso del sangue pellet cellulare (Figura 1). Se queste bande non sono chiare, il processo di separazione non era pulito e dovrà essere ripetuto. </li><li> Rimuovere con attenzione i primi tre strati (plasma, monociti, e terreni di isolamento) usando una pipetta. Smaltire questi strati. </li><li> Con attenzione pipetta lo strato di neutrofili e di tutti i terreni di isolamento sotto il neutrofili. Mettere la soluzione in una provetta da centrifuga pulita. </li><li> Diluire la soluzione dei neutrofili a 10 ml con HBSS senza Ca 2 + / Mg 2 +. Invertire il tubo di un paio di volte a sospendere le cellule. </li><li> Centrifugare la soluzione dei neutrofili a 350 RCF per 10 minuti. Una pallina rossa deve essere presente sul fondo della provetta, contenente residui di neutrofili e globuli rossi (RBC). Eliminare il surnatante con una pipetta attenzione in modo che il pellet non è disturbato. </li><li> Per lisare i globuli rossi residuo, aggiungere 2 ml di Lysis Buffer Red Cell per il tubo. Per risospendere il, pellet vortice del flacone in un ambiente di 3-4. Evitare di aumentare l&#39;impostazione vortice superiore a 4, dal momento che questo può causare i neutrofili da attivare. Potrebbe essere necessario vortex per alcuni secondi, o di &quot;impulso&quot; il vortice di sciogliere il pellet. </li><li> Centrifugare la provetta a 250 RCF per 5 min. Togliere il surnatante con la pipetta. Ripetere il processo di lisi, se necessario. </li><li> Aggiungere 500 microlitri HBSS senza Ca 2 + / Mg 2 + ad ogni provetta. Ancora una volta, vortice per risospendere il pellet in un ambiente di 3-4. Diluire a 10 ml con HBSS senza Ca 2 + / Mg 2 +. </li><li> Centrifugare le provette a 250 RCF per 5 min. Aspirare il surnatante e gettarlo. </li><li> Risospendere il pellet in 250 microlitri HBSS / Soluzione HSA (2% HSA). Le cellule possono essere contati e regolato concentrazione desiderata. </li></ol>

<ol>
	<li>Boyum, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separation+of+leucocytes+from+blood+and+bone+marrow.">Separation of leucocytes from blood and bone marrow.</a> Scand. J. Clin. Invest. 21, 77-83 (1968).</li><li>England, J. M., Rowan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+assignment+of+values+to+fresh+blood+used+for+calibrating+automated+blood+cell+counters.">The assignment of values to fresh blood used for calibrating automated blood cell counters.</a> Clin. Lab. Haemat. 10, 203-212 (1988).</li><li>Garcia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+two+leukocyte+extraction+methods+for+cytomegalovirus+antigenemia+assay.">Comparison of two leukocyte extraction methods for cytomegalovirus antigenemia assay.</a> J. Clin. Microbiol. 34, 182-184 (1996).</li><li>Ferrante, A., Thong, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+conditions+for+simultaneous+purification+of+mononuclear+and+polymorphonuclear+leucocytes+from+human+peripheral+blood+by+the+Ficoll-Hypaque+method.">Optimal conditions for simultaneous purification of mononuclear and polymorphonuclear leucocytes from human peripheral blood by the Ficoll-Hypaque method.</a> J. Immunol. Methods. 36, 109-109 (1980).</li></ol>

Il gradiente di densità metodo di separazione viene utilizzato per isolare neutrofili umani da sangue intero con una miscela di sodio metrizoate e Destrano 500. Questo metodo si basa sul metodo di separazione dei leucociti mononucleari da Boyum (1968), che è stato modificato per la separazione dei neutrofili da Ferrante e Thong (1980). Dopo la raccolta di un donatore, il sangue intero può essere anticoagulato con EDTA, citrato o eparina. Dal momento che sono di breve durata, i neutrofili devono essere utilizzati entro 2-4 ore dalla raccolta. La procedura consiste di sangue intero stratificazione nel medio gradiente di densità, centrifugazione, separazione dello strato dei neutrofili, e lisi degli eritrociti residui. Le cellule vengono poi lavate, contate e risospese alla concentrazione desiderata. Se sei bande non sono distinte dopo la prima fase di centrifugazione, il processo di separazione non è stata pulita e dovrà essere ripetuto. Per la netta separazione, assicurarsi che i terreni di isolamento non è scaduto o contaminato. La separazione può anche fallire se il donatore di sangue ha consumato alcol o farmaci nelle 72 ore precedenti il ​​prelievo del sangue. Per evitare l&#39;attivazione dei neutrofili durante la procedura di separazione, è meglio usare HBSS senza Ca2 + / Mg2 +, dal momento che gli ioni hanno dimostrato di cellule primo. Risospensione del pellet deve essere eseguita lentamente, e le impostazioni del vortice dovrebbe essere tenuto in basso a medio raggio in modo che le cellule non si attivano. Quando eseguito correttamente, questo metodo ha dimostrato di produrre campioni di&gt; neutrofili 95% con vitalità&gt; 95%. La purezza e la vitalità può essere valutata mediante l&#39;etichettatura le cellule con neutrofili specifico marcatore CD66b e trypan esclusione del colorante blu.

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<td>Lymphocyte Poly(R)</td>
<td>Reagent</td>
<td>Cedarlane Labs</td>
<td>&nbsp;</td>
<td>PolymorphprepTM can be used as an alternative</td>
</tr>
<tr>
<td>Hank's Balanced Salt Solution without Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Human Serum Albumin</td>
<td>Reagent</td>
<td>ZLB Bioplasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Red Cell Lysis Buffer</td>
<td>Reagent</td>
<td>Roche Diagnostics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Centrifuge</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Vortexer</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>PolymorphprepTM</td>
<td>Reagent</td>
<td>Axis-Shield PoC</td>
<td>&nbsp;</td>
<td>Alternative reagent to Lymphocyte-Poly (R)</td>
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<tr>
<td>Syringe Filter</td>
<td>Other</td>
<td>Millipore</td>
<td>SLGV033RS</td>
<td>Millex-GV, 0.22 &#181;m, PVDF, 33 mm, gamma-sterilizable</td>
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<tr>
<td>Hank's Balance Salt Solution with Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
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neutrophil isolation protocol

Granulociti neutrofili polimorfonucleati (PMN) sono i leucociti più abbondanti negli esseri umani e tra le prime cellule ad arrivare sul luogo del infiammatoria risposta immunitaria. A causa del loro ruolo chiave nel processo infiammatorio, le funzioni dei neutrofili come locomozione, la produzione di citochine, fagocitosi, e combattere cellule tumorali sono ampiamente studiati. Per caratterizzare le funzioni specifiche di neutrofili, un metodo pulito, veloce e affidabile che li separa dai globuli altro è auspicabile che studi in vitro, specialmente da quando i neutrofili sono di breve durata e deve essere utilizzato entro 2-4 ore dalla raccolta. Qui, dimostrare un metodo standard di gradiente di densità di separazione per isolare neutrofili umani da sangue intero utilizzando mezzi di separazione disponibile in commercio che è una miscela di sodio metrizoate e Destrano 500. La procedura consiste di sangue intero stratificazione nel medio gradiente di densità, centrifugazione, separazione dello strato dei neutrofili, e lisi degli eritrociti residui. Le cellule vengono poi lavate, contate e risospese in tampone alla concentrazione desiderata. Quando eseguito correttamente, questo metodo ha dimostrato di produrre campioni di&gt; neutrofili 95% con vitalità&gt; 95%.

Watch this Scientific Journal Video about Neutrophil Isolation Protocol at JoVE.com

Neutrophil Isolation Protocol

I neutrofili sono tra le prime cellule ad arrivare sul luogo del infiammatoria risposta immunitaria, e le loro funzioni e meccanismi sono stati ampiamente studiata in vitro. Si dimostra uno standard gradiente di densità metodo di separazione per isolare neutrofili umani da sangue intero utilizzando mezzi di separazione disponibili in commercio.

Neutrofili isolamento Protocollo

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

Nanyang Technological University

Research

JoVE Journal

Biology

101.8K Views.  University of Pennsylvania. I neutrofili sono tra le prime cellule ad arrivare sul luogo del infiammatoria risposta immunitaria, e le loro funzioni e meccanismi sono stati ampiamente studiata in vitro. Si dimostra uno standard gradiente di densità metodo di separazione per isolare neutrofili umani da sangue intero utilizzando mezzi di separazione disponibili in commercio.

Video: Neutrophil Isolation Protocol

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

Protocollo per la cura dei Mosquito (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

Ricerca

Biologia

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescenza In situ</emIbridazione> (FISH) Protocollo di sperma umano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

Un protocollo per la produzione di KLRG1 tetramero

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

Basati sul DNA Fish Identification Specie protocollo

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

SIVQ-LCM protocollo per lo strumento ArcturusXT

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Protocollo per l&#39;isolamento di epatociti umani primari e corrispondenti principali popolazioni di cellule del fegato non-parenchimali

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

Protocollo di controllo dell&#39;orologio per l&#39;analisi delle immagini: plugin ImageJ

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Un semplice protocollo ad alta efficienza per l'isolamento delle isolozioni pancreatiche dai topi

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high concentrations in specific and tertiary granules of neutrophils. This protocol details how MUB40&#160;conjugated directly to a fluorophore can be used to stain neutrophils in fixed/permeabilized tissues as well as how this can be used in live-cell imaging to assay for neutrophil activation and de-granulation. Neutrophil detection methods are limited to species-specific monoclonal antibodies, which are not always suitable for certain applications. MUB40 does not penetrate the cell membrane and is thus excluded from lactoferrin stored in non-activated/non-permeabilized neutrophils. MUB40 has the added benefit of recognizing lactoferrin from a broad host range, making it especially useful for comparing results in studies involving multiple research models, reducing the number of duplicate reagents, and simplifying protocols through single-step staining.

The MUB40 Peptide for Use in Detecting Neutrophil-Mediated Inflammation Events

Here, we present a protocol to detect the presence of neutrophils in fixed/permeabilized histology sections and assess the activation state of live purified neutrophils. In particular, the MUB40&#160;peptide binds lactoferrin present in neutrophil-specific and tertiary granules. Exposure of the granule contents through either permeabilization or neutrophil activation allows for the marking of neutrophils.

Il Peptide di40 MUB per uso nella rilevazione eventi infiammatori mediato dai neutrofili

Here, we provide a protocol involving the use of MUB40, a synthesized peptide with the ability to bind glycosylated lactoferrin stored at high ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Protocollo di isolamento gocciolina del lipido rapida utilizzando un Kit di isolamento organello ben consolidate

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...