资金从美国国立卫生研究院R01 HL56621。

嗜中性粒细胞的分离协议<ol><li>将所有试剂置于室温。 </li><li>收集的5.0毫升离心管中的中性粒细胞隔离媒体。仔细层5.0毫升的血液分离介质，。执行这一步，缓慢而仔细地，并与介质的表面，以避免混合血液和媒体的枪头。 </li><li> 500 35分钟的离心力离心20-25℃。血液分离出6个不同组别：血浆，单核细胞，隔离媒体，中性粒细胞，更加孤立的媒体，以及红血细胞沉淀（图1）。如果不明确，这些频段的分离过程是不干净，将需要重复。 </li><li>小心地取出前三名层（血浆，单核细胞，并进行隔离媒体），用吸管。这些层的处理。 </li><li>仔细吸管嗜中性粒细胞和中性粒细胞下方的隔离媒体所有的层。放置到一个干净的离心管的解决方案。 </li><li>没有的Ca 2 + / Mg 2 +的HBSS中稀释的中性粒细胞10毫升的解决方案。反转管几次暂停细胞。 </li><li>离心机的中性粒细胞在10分钟的350离心力的解决方案。一个红色的沉淀应在试管底部，包含中性粒细胞和残留的红血细胞（红细胞）。用移液器取出上清液小心，颗粒不被打扰。 </li><li>裂解残留的红细胞，加入2 ml红细胞裂解液管。悬浮颗粒，旋涡在小瓶设置3-4。避免增加上述4旋涡设置，因为这可能会导致中性粒细胞激活。它可能是必要的，以涡几秒钟，或以“脉冲”涡旋溶解沉淀。 </li><li> 250 5分钟的离心力离心管。取出上清液用吸管。重复裂解过程中，如果需要的话。 </li><li>没有的Ca 2 + / 镁加500μL的HBSS +每管。同样，旋涡沉淀重悬在设置3-4。没有的Ca 2 + / Mg 2 +的HBSS中，稀释至10 ml 。 </li><li> 250 5分钟的离心力离心管。吸出上清液并丢弃。 </li><li> 250μL的HBSS / HSA的解决方案（2％，HSA）重悬沉淀。然后可以计算细胞，并调整到所需浓度。 </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>

密度梯度分离法是用来隔离从全血混合使用metrizoate钠和右旋糖酐500人中性粒细胞。这种方法是基于对Boyum（1968年）的单核白细胞分离法修改费兰特和通（1980）中性粒细胞分离。 从捐赠者收集后，全血用EDTA，柠檬酸或肝素抗凝。由于它们是短命的，中性粒细胞应收集2-4小时内使用。由分层全血通过离心，密度梯度介质，中性粒细胞层的分离，剩余红​​细胞裂解的过程。细胞，然后洗净，计算，并重新悬?...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<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>
<tr>
<td>Human Serum Albumin</td>
<td>Reagent</td>
<td>ZLB Bioplasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Red Cell Lysis Buffer</td>
<td>Reagent</td>
<td>Roche Diagnostics</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Vortexer</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Pipettes and bulb</td>
<td>Tool</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PolymorphprepTM</td>
<td>Reagent</td>
<td>Axis-Shield PoC</td>
<td>&nbsp;</td>
<td>Alternative reagent to Lymphocyte-Poly (R)</td>
</tr>
<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>
</tr>
<tr>
<td>Hank's Balance Salt Solution with Calcium Chloride</td>
<td>Reagent</td>
<td>Invitrogen Gibco</td>
<td>&nbsp;</td>
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neutrophil isolation protocol

中性粒细胞的多形核粒细胞（PMN）在人类之间的第一个细胞到达炎症免疫反应的部位，最丰富的白细胞。由于其关键作用，在炎症，如中性粒细胞功能的运动，细胞因子的产生，的吞噬功能，和肿瘤细胞的作战广泛的研究。为特征的中性粒细胞的特定功能，其他血细胞分离干净，快速，可靠的方法是可取的，尤其是因为在体外研究中性粒细胞是短命的，应在2-4小时收集。在这里，我们展示了一个标准的密度梯度分离法，从全血使用市售分离介质，是一种混合物，钠metrizoate和右旋糖酐500人中性粒细胞隔离。由分层全血通过离心，密度梯度介质，中性粒细胞层的分离，剩余红​​细胞裂解的过程。细胞，然后洗净，计数，并在缓冲区所需浓度的悬浮。正确执行时，此方法已被证明收益率&gt; 95％嗜中性粒细胞&gt; 95％的可行性的样品。

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

Neutrophil Isolation Protocol

嗜中性粒细胞之间的第一个细胞到达炎症免疫反应的工地上，和其职能和机制，已被广泛地在体外研究。我们展示一个标准密度梯度分离法，隔离人类使用市售的分离介质从整个血液中性粒细胞。

嗜中性粒细胞的分离协议

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

Research

JoVE Journal

Biology

101.7K 次观看.  University of Pennsylvania. 嗜中性粒细胞之间的第一个细胞到达炎症免疫反应的工地上，和其职能和机制，已被广泛地在体外研究。我们展示一个标准密度梯度分离法，隔离人类使用市售的分离介质从整个血液中性粒细胞。

视频: 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.

蚊子养育的议定书“（冈比亚按蚊）

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 ...

科研

生物学

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.

以DNA为基础的鱼类物种鉴定协议

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

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.

协议主要人肝细胞的分离和相应的非实质肝细胞的主要种群

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

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.

胰腺胰岛与小鼠分离的简单高效协议

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.

mub40肽在中性炎症事件检测中的应用

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.

利用成熟的细胞隔离试剂盒快速分离脂滴器

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

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

一种改进的小鼠原代肝细胞分离时间和劳动效率方案

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...

Imaging Neutrophil Migration in the Mouse Skin to Investigate Subcellular Membrane Remodeling Under Physiological Conditions

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first immune cells to reach the site of inflammation and to participate in the innate immune response during infection or tissue damage. So far, neutrophil migration has been successfully visualized using various in vitro experimental systems based on uniform stimulation, or confined migration under agarose, or micro-fluidic channels. However, these models do not recapitulate the complex microenvironment that neutrophils encounter in vivo. The development of multiphoton microscopy (MPM)-based techniques, such as intravital subcellular microscopy (ISMic), offer a unique tool to visualize and investigate neutrophil dynamics at subcellular resolutions under physiological conditions. In particular, the ear of a live anesthetized mouse provides an experimental advantage to follow neutrophil interstitial migration in real-time due to its ease of accessibility and lack of surgical exposure. ISMic provides the optical resolution, speed, and depth of acquisition necessary to track both cellular and, more importantly, subcellular processes in 3D over time (4D). Moreover, multi-modal imaging of the interstitial microenvironment (i.e., blood vessels, resident cells, extracellular matrix) can be readily accomplished using a combination of transgenic mice expressing select fluorescent markers, exogenous labeling via fluorescent probes, tissue intrinsic fluorescence, and second/third harmonic generated signals. This protocol describes 1) the preparation of neutrophils for adoptive transfer into the mouse ear, 2) different settings for optimal sub-cellular imaging, 3) strategies to minimize motion artifacts while maintaining a physiological response, 4) examples of membrane remodeling observed in neutrophils using ISMic, and 5) a workflow for the quantitative analysis of membrane remodeling in migrating neutrophils in vivo.

Neutrophil migration relies on the rapid and continuous remodeling of the plasma membrane in response to chemoattractant and its interactions with the extracellular microenvironment. Described herein is a procedure based on Intravital Subcellular Microscopy to investigate the dynamic of membrane remodeling in neutrophils injected in the ear of anesthetized mice.

对小鼠皮肤中的中性粒细胞迁移进行成像，以研究生理条件下的亚细胞膜重塑

The study of immune cell recruitment and function in tissues has been a very active field over the last two decades. Neutrophils are among the first ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

通过 CLON-G 和 体外 自发死亡测定延长中性粒细胞寿命

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

从冷冻哺乳动物组织中分离和分析单个细胞核

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

作者聚焦：增强药物发现 - 开发从冷冻组织中提取细胞核的自动化标准化方案 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.