Biz Dr. Naidenko refolding koşulları optimize yardım için teşekkür ederim. Bu çalışma, Ulusal Sağlık Enstitüsü, araştırma hibe AI58181 tarafından desteklenen Laurent Brossay. Cindy Banh NIH NRSA F31 AI080230 tarafından desteklenmektedir. Stephanie Terrizzi ve Cindy Banh bu çalışmaya katkıda bulunur.

İçerme Organları Hazırlanması <ol><li> TB 4 x 500 ml&#39;lik şişeler İnokülasyon her bir gecede KLRG1 plazmid inşa ifade bakteri kültürü ile 50 mcg / ml carbenicillin ve kloramfenikol içeren. OD 600 nm dalga boyunda 0.6 Å ulaştığında, 37 ilavesi ile 0.4 M IPTG neden ve kültür sallayarak ek bir 4 saat inkübe ° C </li><li> Hasat edilen hücrelerin tabanda tampon pH = 8.0 (50 mM Tris-HCl,% 25 (W / V), sukroz, 1 mM EDTA, 10 mM DTT) süspanse edin. Not: Bu aşamada pelet dondurulabilir. </li><li> Polipropilen beher bakteriyel resuspensions en fazla 60 ml Havuzu. Gerekli TE tamponu, pH 8.0 ile 60 ml ayarlamak ve karışımı yarım hızda karıştırın. </li><li> Karışımı karıştırarak için açılan bilge ekleyin: lizozim (son = 1 mg / ml), MgCl2 (son = 5 mM), 2 mg DNAz Ben 75 mM NaCl, Triton-X100 (son =% 1), DTT (% 50 gliserol içerdiği son = 10 mM). </li><li> 1.5 dakika süreyle buz üzerinde çözüm sonikasyon ve 10 dakika için 5,000 RPM lizatları santrifüj 4 ° C <ol class="lalpha"><li> Süpernatant Durusu. </li><li> 15 ml yıkama tamponu pH = 8.0 (50 mM Tris-HCl,% 0.5 Triton X-100, 100 mM NaCl, 1 mM EDTA, 1 mM DTT). </li><li> Pelet tamamen yeniden süspanse kadar buz üzerinde, 1.5 dakika için çözüm sonikasyon. </li><li> 4 10 dakika için 5,000 RPM örnekleri Santrifüj ° C </li><li> Yıkama 5 kez tekrarlayın. </li></ol></li><li> TE Tampon 4 ml Triton X-100, santrifüj ve tekrar süspansiyon olmadan yıkama tamponu ile 5b tekrarlayın. </li><li> 30 ila 100 mg / ml konsantrasyonda ıslak ağırlık dahil organları bulamacı ve TE tampon mağaza alın. </li></ol> KLRG1, Refolding <ol><li> Tampon pH refolding 1 L = 8.0 (0.4 M Arginin-HCl, 0,1 M Tris pH 8-8,3, 2 mM EDTA, 0.2 mM PMSF, 3 EDTA-proteaz inhibitörü tablet, 5 mM GSH ve 0.5 mM GSSG) ve soğuk 4 ° C karıştırma iken. </li><li> Dahil organları katlama karışımın içine enjeksiyon için hazırlayın: protein konsantrasyonu son 2 3 mcM böylece her 5 10 0.25 0.5 mcM konsantrasyon ile enjeksiyon yapmak için idealdir. <ol class="lalpha"><li> 7 M GnHCl dahil organları bulamaç 50 mg hacmi 10 mM β-mercaptoethanol eritin. </li><li> Tutun 37 dahil organları / GnHCl karışımı ° C için 30 - 40 dakika ve vorteks her 5 dakikada tam bir erime sağlamak için (bulamaç tamamen erimiş olacak). </li><li> 4 mikrosantrifüj 30 dakika maksimum hızda Santrifüj ° C ve 15 ml santrifüj tüpüne süpernatantı aktarın. Küçük siyahımsı pelet herhangi bir transfer değil. </li></ol></li><li> Enjeksiyon tampon (3 M GnHCl, 10 mM NaAcetate pH 4.2,10 mM EDTA) ile ses seviyesini ayarlayın. </li><li> 1 ml seyreltilmiş dahil organları her saat enjekte edilir. Enjekte zaman, yüksek hızda karıştırın 2 saniye arasında her damla damla 1 ml seyreltilmiş dahil organları damla ekleyin. Enjeksiyonu yapıldığında, düşük hızda karıştırın. </li><li> 4 düşük hızda karıştırarak gecede Devam ° C </li></ol> Refolding Reaksiyonlar konsantrasyonu <ol><li> Millipore s protokolünü takiben, bir Amicon kullanarak önceden filtrelenmiş (0.22 mikron filtreli) refolding reaksiyon 1 L konsantre Hücre ve ~ 10 ml YM10 NMWL 10K ultrafiltrasyon membran (Millipore) Karıştırmalı. </li><li> ≤ 2 ml Konsantre bir Centriplus YM-10 10K MWCO (Millipore). </li></ol> KLRG1 tetramer saflaştırılması <ol><li> 4 AKTAFPLC Kur ° C GE Healthcare kılavuzları göre. </li><li> Boyut dışlama kromatografisi ile bir Sephacryl S-200, 16/60, yüksek çözünürlüklü 20 mM HEPES sütun (GE Healthcare) ve 150 mM NaCl kullanarak KLRG1 monomer formu arındırın. (Bakınız Şekil 1). </li><li> Centriplus kullanarak 1 ml Konsantre YM-10 10K MWCO (Millipore). </li><li> KLRG1 monomer biotinylate Avidite s protokole göre Bira enzim kullanın. </li><li> Serbest biotin 2 boyut dışlama kromatografisi ile elimine edilir. </li><li> KLRG1 molekülü KLRG1 monomer 4 katlı molar aşırı PE konjuge streptavidin (BD Biosciences) ile karıştırılarak tetramerized. </li></ol> Temsilcisi Sonuçlar <a href="https://www.jove.com/files/ftp_upload/1702/1702fig1.jpg"><img src="/files/ftp_upload/1702/1702fig1tmb.jpg" alt="Şekil 1" border="0" /></a> Şekil 1 AKTA FPLC boyut dışlama kromatografisi. Temsilcisi olumlu sonuçlar KLRG1 refolded. Monomer ayırma (83,52 ml), dimer (56,36 ml) ve refolded KLRG1 multimer (36,26 ml) form grafiğini gösterir. 94,25 ml zirve tampon değişimi temsil eder.

<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>

Bu protokol, bu biotinylate arındırmak için nasıl gösterildiği ve tetramerize KLRG1. KLRG1 tetramer flow sitometri KLRG1 ligandlar etiketlemek için kullanılabilir. Refolding koşulları her protein için ampirik olarak test edilmesi olsa da, diğer C-tipi lektin benzer bir protokol kullanılarak tetramerized olabilir. Problar, C-tipi lektin reseptörleri yetim ligandlar olarak tetramerized proteinleri kullanarak potansiyel olarak tespit edilebilir.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<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

Öldürücü hücre lektin-benzeri reseptör G1 (KLRG1) C tipi lektin-benzeri süperailesinin ait bir tip II transmembran glikoprotein inhibitör reseptör. KLRG1 bir monomer olarak ve bir disülfit bağlantılı homodimer olarak var. Bu iyi korunmuş reseptör, en olgun ve en yakın zamanda aktif NK hücreleri gibi efektör / bellek T hücrelerinin bir alt kümesi bulunur. KLRG1 tetramer yanı sıra diğer yöntemleri kullanarak, E, K, ve R-cadherins KLRG1 ligandlar olarak tespit edilmiştir. Bu Ca 2 + bağımlı hücre-hücre adezyon molekülleri, hücre-hücre etkileşimleri, bir transmembran bir etki alanı ve aktin hücre iskeletinin bağlı bir sitoplazmik etki alanı için sorumlu beş kaderin tekrarlar içeren bir ekstrasellüler etki oluşur. KLRG1 tetramer Üretimi KLRG1 ligandlar belirlenmesi için gerekli idi. KLRG1 tetramer immün yanıt ve doku bütünlüğünü düzenleyen rolleri cadherin ve KLRG1 oyun aydınlatmak için de eşsiz bir araç.

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

Bu protokol KLRG1 ligandların analizi için güçlü bir araçtır KLRG1 tetramer, üretim açıklamaktadır.

KLRG1 tetramer Üretim için Protokol

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

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9.9K Görüntüleme.  Brown University. Bu protokol KLRG1 ligandların analizi için güçlü bir araçtır KLRG1 tetramer, üretim açıklamaktadır.

Video: 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.

Sivrisinek Yetiştirme Protokolü (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 ...

Biyoloji

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.

Görselleştirme Antijen spesifik CD4 + T hücreleri MHC Sınıf II Tetramers kullanarak

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.

Üretimi C. elegans Recombineering yoluyla transgenlerin GalK Seçilebilir 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 ...

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.

İnsan Hücrelerden salgılanan Proteinlerin Üretimi için Kullanışlı ve Genel Anlatım Platformu

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.

Vahşi Tırtıllar gelen Hemocytes ayıklamak için basit bir protokol

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.

ArcturusXT Instrument için SIVQ-LCM Protokolü

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.

Ekstraselüler Asit Üretim Ölçüm ve Analiz Glikolitik Hızı belirleme

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.

Probiyotik Fermente Gıdaların küçük ölçekli imalat için yeni Üretim Protokolü

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.

Kas Dokular doğrudan sıvı azot içine girdikleri zaman Artifacts Donma bir çoklu-delikli dondurarak saklamaya uygun vialler ortadan kaldırır

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.