我们感谢博士Naidenko优化复性条件的帮助。这项工作是支持由美国国立卫生研究院的研究经费AI58181洛朗Brossay。辛迪迪班是由美国国立卫生研究院NRSA F31 AI080230支持。 斯蒂芬妮Terrizzi和辛迪迪班平等作出贡献，对这项工作。

包涵体的制备<ol><li>接种4 × 500毫升烧瓶结核病每个过夜培养的细菌，表达KLRG1质粒构建含有50微克/ ml的羧苄青霉素和氯霉素。当外径达到0.6 A，在600 nm与0.4 M IPTG诱导，并在37孵育一个附加的4小时晃动文化° C。 </li><li>中提取的干细胞重悬在再悬浮缓冲液的pH值= 8.0（50 25％（W / V）蔗糖，1 mM的EDTA，MM的Tris - HCl，10 mM的数码地面电视）。注：颗粒可以在这个阶段被冻结。 </li><li>池不超过60毫升细菌resuspensions的聚丙烯烧杯。如果有必要调整到60毫升，用TE缓冲液pH值8.0，并在半速搅拌混合。 </li><li>以搅拌混合添加下降明智的：溶菌酶（终= 1毫克/毫升），氯化镁（终= 5毫米），2毫克DNA酶我包含在50％的甘油75毫米氯化钠，TRITON - X100（最终= 1％），数码地面电视（最终= 10毫米）。 </li><li>超声冰1.5分的解决方案，并在10分钟5000转离心裂解液在4 ° C。 <ol class="lalpha"><li>倾倒上清液。 </li><li>添加15毫升的洗涤缓冲液的pH值= 8.0（50毫米的Tris - HCl，0.5％Triton X - 100的的氯化钠，100毫米，1毫米EDTA，1 mM的数码地面电视）。 </li><li>在冰上，超声1.5分钟的解决方案，直到沉淀完全重悬。 </li><li> 10分钟5000转离心样品在4 ° C。 </li><li>重复洗5次。 </li></ol></li><li>洗没有TRITON X - 100，离心机和4毫升TE缓冲液中的悬浮缓冲液重复5B。 </li><li>以湿重包涵体浆在30至100毫克/毫升的浓度，并存储在TE缓冲液。 </li></ol>复性KLRG1 <ol><li> 1 L复性缓冲液pH = 8.0（0.4 M精氨酸盐酸的0.1 M Tris pH值8-8.3，2毫米EDTA，0.2毫米PMSF，EDTA - 免费蛋白酶抑制剂药片，5毫米GSH和0.5毫米的GSSG）和寒意到4 ° C，边搅拌。 </li><li>准备注入折叠混合物包涵体：这是非常理想，使每5 10注射用0.25 0.5微米的浓度，使终浓度的蛋白质将于2 3微米。 <ol class="lalpha"><li>融化10毫米的β-巯基乙醇50毫克包涵体浆量在7米GnHCl。 </li><li>保持包涵体/ GnHCl混合物在37 ° C为30 - 40分钟和涡每5分钟，以确保完全融化（完全融化时，泥浆就会清楚）。 </li><li>离心机最大速度离心30分钟在4 ° C，将上清转移至15 mL离心管。做不转让任何黑色的小颗粒。 </li></ol></li><li>注射缓冲液（3米GnHCl，10毫米NaAcetate mM的EDTA，pH值4.2,10），调整音量。 </li><li>注入1毫升稀释后的包涵体每隔一小时。注射时，在高速搅拌，添加1毫升稀释包涵体下降，下降2秒之间每下降。当注射完成后，在低速搅拌。 </li><li>隔夜继续低速搅拌，在4 ° C。 </li></ol>浓度的复性反应<ol><li> Millipore公司S协议，集中1升的预过滤（0.22μm的过滤），复性反应使用Amicon搅拌细胞和YM10 NMWL 10K超滤膜（Millipore公司）〜10毫升。 </li><li>集中≤2毫升，使用一个Centriplus YM - 10 10K截留分子量（Millipore公司）。 </li></ol> KLRG1四聚体的纯化<ol><li>安装在4 ° C，符合GE医疗手册的AKTAFPLC。 </li><li>使用Sephacryl S - 200 16/60高分辨率柱（通用电气医疗集团）在20毫米的HEPES和150 mM氯化钠尺寸排阻色谱纯化单体形式存在的KLRG1。 （见图1）。 </li><li>浓缩至1 ml，使用Centriplus YM - 10 10K截留分子量（Millipore公司）。 </li><li>根据以亲和力S协议，使用比拉酶biotinylate KLRG1单体。 </li><li>免费素是体积排阻色谱2淘汰。 </li><li> KLRG1分子tetramerized KLRG1单体与4倍超额摩尔聚乙烯共轭链霉亲和素（BD Biosciences公司）混合。 </li></ol>代表性的成果 <a href="https://www.jove.com/files/ftp_upload/1702/1702fig1.jpg"><img src="/files/ftp_upload/1702/1702fig1tmb.jpg" alt="图1" border="0" /></a> AKTA FPLC尺寸排阻色谱图1。代表从积极的成果复性KLRG1。图表显示分离（83.52毫升）的单体，二聚体（56.36毫升），和multimer（36.26毫升）的复性KLRG1形式。 94.25毫升峰代表缓冲区交换。

<ol>
	<li>Tessmer, M. S., Fugere, C., Stevenaert, F., Naidenko, O. V., Chong, H. J., Leclercq, G., Brossay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KLRG1+binds+cadherins+and+preferentially+associates+with+SHIP-1.">KLRG1 binds cadherins and preferentially associates with SHIP-1.</a> Int Immunol. 19, 391-400 (2007).</li><li>Grundemann, C., Bauer, M., Schweier, O., von Oppen, N., Lassing, U., Saudan, P., Becker, K. F., Karp, K., Hanke, T., Bachmann, M. F., Pircher, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+identification+of+E-cadherin+as+a+ligand+for+the+murine+killer+cell+lectin-like+receptor+G1.">Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.</a> J Immunol. 176, 1311-1315 (2006).</li><li>Ito, M., Maruyama, T., Saito, N., Koganei, S., Yamamoto, K., Matsumoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killer+cell+lectin-like+receptor+G1+binds+three+members+of+the+classical+cadherin+family+to+inhibit+NK+cell+cytotoxicity.">Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity.</a> J Exp Med. 203, 289-295 (2006).</li><li>Li, Y., Hofmann, M., Wang, Q., Teng, L., Chlewicki, L. K., Pircher, H., Mariuzza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+natural+killer+cell+receptor+KLRG1+bound+to+E-cadherin+reveals+basis+for+MHC-independent+missing+self+recognition.">Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.</a> Immunity. 31, 35-46 (2009).</li><li>Nakamura, S., Kuroki, K., Ohki, I., Sasaki, K., Kajikawa, M., Maruyama, T., Ito, M., Kameda, Y., Ikura, M., Yamamoto, K., Matsumoto, N., Maenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K.+Molecular+basis+for+E-cadherin+recognition+by+killer+cell+lectin-like+receptor+G1+(KLRG1).">K. Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).</a> J Biol Chem. , .</li></ol>

在这个协议中，它是如何净化，biotinylate，tetramerize KLRG1。 KLRG1四聚体可以用来标记流式细胞仪KLRG1配体。虽然复性条件进行测试，每个蛋白质的经验，其他的C型凝集素可tetramerized使用了类似的协议。使用tetramerized蛋白作为探针，对孤儿的C型凝集素受体的配体可能被确定。

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>BirA Enzyme and Buffer</td>
<td>Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitor Tablets, EDTA Free</td>
<td>Reagent</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>or equivalent</td>
</tr>
<tr>
<td>Amicon Stirred Cell</td>
<td>Equipment</td>
<td>Millipore</td>
<td>Model #8400</td>
<td>or equivalent</td>
</tr>
<tr>
<td>YM10 NMWL 10K ultrafiltration membrane</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>13642</td>
<td>&nbsp;</td>
<tr>
<td>15 ml YM10 concentrator</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>4411</td>
<td>&nbsp;</td>
<tr>
<td>AKTAFPLC</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>18-1900-26</td>
<td>&nbsp;</td>
<tr>
<td>Sephacryl S-200 16/60 high-resolution column</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>17-1166-01</td>
<td>&nbsp;</td>
<tr>
<td>Strepavidin PE</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>554061</td>
<td>or equivalent</td>
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a protocol for the production of klrg1 tetramer

杀伤细胞凝集素样受体G1（KLRG1）是II型跨膜糖蛋白抑制性受体，属于C型凝集素样超家族。 KLRG1存在单体和二硫键相连的二聚体。这种保守的受体上最成熟，最近激活NK细胞，以及效应/记忆性T细胞的一个子集。 使用KLRG1四聚体，以及其他的方法，E，N，和R -钙粘素被确定为KLRG1配体。这些CA 2 +依赖的细胞，细胞粘附分子由胞外域包含五个cadherin的细胞间的相互作用，跨膜结构域和细胞质域与肌动蛋白细胞骨架的重复。 KLRG1四聚体的生成，是必不可少的KLRG1配体的识别。 KLRG1四聚体，也是一个独特的工具，以澄清在调节免疫反应和组织的完整性的作用cadherin和KLRG1发挥。

Watch this Scientific Journal Video about A Protocol for the Production of KLRG1 Tetramer at JoVE.com

A Protocol for the Production of KLRG1 Tetramer

这个协议描述KLRG1四聚体的生产，这是一个强大的工具KLRG1配体的分析。

KLRG1四聚体的生产协议

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

a-protocol-for-the-production-of-klrg1-tetramer

Research

JoVE Journal

Biology

9.9K 次观看.  Brown University. 这个协议描述KLRG1四聚体的生产，这是一个强大的工具KLRG1配体的分析。

视频: A Protocol for the Production of KLRG1 Tetramer

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

Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

可视化抗原特异性CD4 + T细胞的MHC II类四聚四

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

生产 C。线虫</em通过重组工程与转基因 galK选择标记

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

一个从人体细胞分泌蛋白生产的方便，一般的表达平台

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

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.

为ArcturusXT仪器SIVQ-LCM协议

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

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

对于益生菌发酵食品的小规模生产新生产协议

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

的多孔冷冻管省去冻结工件时肌肉组织在液氮中直接浸入

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...

A Protocol for Laboratory Housing of Turquoise Killifish (Nothobranchius furzeri)

The development of husbandry practices in non-model laboratory fish used for experimental purposes has greatly benefited from the establishment of reference fish model systems, such as zebrafish and medaka. In recent years, an emerging fish &#8211; the turquoise killifish (Nothobranchius furzeri) &#8211; has been adopted by a growing number of research groups in the fields of biology of aging and ecology. With a captive life span of 4 - 8 months, this species is the shortest-lived vertebrate raised in captivity and allows the scientific community to test &#8211; in a short time &#8211; experimental interventions that can lead to alterations of the aging rate and life expectancy. Given the unique biology of this species, characterized by embryonic diapause, explosive sexual maturation, marked morphological and behavioral sexual dimorphism - and their relatively short adult life span -&#160;ad hoc husbandry practices are in urgent demand. This protocol reports a set of key husbandry measures that allow optimal turquoise killifish laboratory care, enabling the scientific community to adopt this species as a powerful laboratory animal model.

A Protocol for Laboratory Housing of Turquoise Killifish Nothobranchius furzeri

Laboratory housing of turquoise killifish can be scaled up&#160;to house and efficiently raise thousands of individual fish in a centralized water filtration system, employing the same infrastructure used for standard zebrafish facilities. Here we detail a list of standardized procedures that allow efficient killifish maintenance.