This work was partly supported by CSIC (I+D project C022) and PEDECIBA. The authors want to thank Mariela Bollati and Valentina Porro from the Cell Biology Unit (Institut Pasteur de Montevideo) for their generous collaboration concerning the MoFlo cell sorter, and Merial-Montevideo for gently providing all the guinea pig specimens used in this project. Figure 1 was reproduced with permission of BioMed Central; Figures 2 and 3, and Table 1 with permission of S. Karger AG, Basel; and Figures 4 and 5 were reproduced or adapted with permission of John Wiley &amp; Sons, Inc (copyright owned by Wiley-Blackwell, 2011).

1.	Preparation of Cell Suspensions
<ol>
 <li>Sacrifice the specimen to be used following the recommendations of the specialized committees such as IACUC or equivalent (in Uruguay, National Commission for Animal Experimentation [CNEA]). In our case, an overdose of pentobarbital was administrated. </li>
 <li>Dissect the testes following standard approved procedures and place them in a 96 mm glass Petri dish on ice, containing 10 ml of ice-cold DMEM supplemented with 10% fetal calf serum. </li>
 <li>Remove the tunica albuginea and cut the decapsulated testes into square pieces of 2 - 3 mm on each side. </li>
 <li>Those pieces are then processed in a Medimachine, an automated mechanical grinder where the tissue is disaggregated inside a disposable unit containing a perforated stainless-steel screen and a metal rotor. To do so, place 1 ml of cold supplemented DMEM and 4 - 5 of these pieces in a 50 &mu;m disposable unit, switch on the disaggregator, and process for 50 sec following the simple instructions from the manufacturer. </li>
 <li>Recover the resulting cell suspension from the disaggregation unit using a 3 - 5 ml syringe without needle. </li>
 <li>Filter through a 50 &mu;m nylon mesh, previously soaked with 0.5 ml supplemented DMEM. </li>
 <li>Filter the suspension again using a soaked 25 &mu;m nylon mesh, and place on ice. </li>
 <li>Count in a Neubauer chamber and adjust cellular concentration to 1 - 2 x 107 cells/ml with supplemented DMEM. At least 4 x 107 cells/gram of testis material are usually obtained. </li>
 <li>Finally, add NDA (2-naphthol-6,8-disulfonic acid, dipotassium salt) to a final concentration of 0.2% in order to prevent cell clumping. </li>
 <li>Optional: check cell viability of the testicular cell suspensions with a commercially available viability kit for animal cells, following manufacturer's instructions. </li>
</ol>
2.	Flow Cytometric Analysis
	We have used a Becton-Dickinson FACSVantage flow cytometer equipped with a Coherent argon ion laser tuned to emit at 488 nm for the analysis of cells stained with high concentrations of propidium iodide (PI). (The issue of PI entrance into unfixed cells under stress has been addressed elsewhere9,10).
<ol>
 <li>For PI staining, add fluorochrome at a final concentration of 50 &mu;g/ml to the cell suspension, and incubate for 10 min at 0 &deg;C in the dark.</li>
 <li>Laser power is set to 100 mW and a 575/26 band pass filter is used to collect PI-emitted fluorescence in FL2. </li>
 <li>We perform FC measurements with a 70 &mu;m nozzle. For sorting spermatocyte populations, set sorting mode in Normal-R or Normal-C, using 3 sorted drops as envelope. Keep sample and collection tubes at 3 - 4 &deg;C by using a refrigeration unit. Adjust sample differential to analyze cells at a rate of 500 to 1,500 per second. </li>
 <li>Use CellQuest software (BD) to analyze the following parameters: forward scatter (FSC-H); side scatter (SSC-H); total emitted fluorescence or pulse-area (FL2-A); and duration of fluorescence emission or pulse-width (FL2-W). </li>
</ol>
Alternatively, we have employed the vital dye Hoechst 33342 to a final concentration of 5 &mu;g/ml and incubated for 10 min at 37 &deg;C in the dark. Cell analysis was performed by means of a MoFlo Cytometer (DakoCytomation) equipped with a UV excitation wavelength laser operating at 25 mW and using a 70 &mu;m nozzle. Data manipulation was performed with Summit v4.3 software (DakoCytomation).

<ol>
	<li>Meistrich, M. L., Prescott, D. M. <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+spermatogenic+cells+and+nuclei+from+rodent+testes.">Separation of spermatogenic cells and nuclei from rodent testes.</a> Methods of Cell Biology. 15, 15-54 (1977).</li><li>Lam, D. M. K., Furrer, R., Bruce, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+separation,+physical+characterization,+and+differentiation+kinetics+of+spermatogonial+cells+of+the+mouse.">The separation, physical characterization, and differentiation kinetics of spermatogonial cells of the mouse.</a> Proc. Natl. Acad. Sci. USA. 65, 192-199 (1970).</li><li>Romrell, L. J., Bellve, A. R., Fawcet, D. W. <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+mouse+spermatogenic+cells+by+sedimentation+velocity.">Separation of mouse spermatogenic cells by sedimentation velocity.</a> Dev. Biol. 19, 119-131 (1976).</li><li>Geisinger, A., Rodr&#237;guez-Casuriaga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+for+gene+expression+studies+in+mammalian+spermatogenesis.">Flow cytometry for gene expression studies in mammalian spermatogenesis.</a> Cytogenet. Genome Res. 128, 46-56 (2010).</li><li>Getun, I. V., Torres, B., Bois, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+purification+of+mouse+meiotic+cells.">Flow cytometry purification of mouse meiotic cells.</a> J. Vis. Exp. (50), e2602 (2011).</li><li>Meistrich, M. L. <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+mouse+spermatogenic+cells+by+velocity+sedimentation.">Separation of mouse spermatogenic cells by velocity sedimentation.</a> J. Cell Physiol. 80, 299-312 (1972).</li><li>Meistrich, M. L., Bruce, W. R., Clermont, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+fractions+of+mouse+testis+cells+following+velocity+sedimentation+separation.">Cellular composition of fractions of mouse testis cells following velocity sedimentation separation.</a> Exp. Cell Res. 79, 213-227 (1973).</li><li>Malkov, M., Fisher, Y., Don, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+schedule+of+the+postnatal+rat+testis+determined+by+flow+cytometry.">Developmental schedule of the postnatal rat testis determined by flow cytometry.</a> Biol. Rep. 59, 84-92 (1998).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., Santi&#241;aque, F., L&#243;pez, B., Folle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-purity+flow+sorting+of+early+meiocytes+based+on+DNA+analysis+of+guinea+pig+spermatogenic+cells.">High-purity flow sorting of early meiocytes based on DNA analysis of guinea pig spermatogenic cells.</a> Cytometry A. 79, 625-634 (2011).</li><li>Davey, H. M., Hexley, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Red+but+not+dead?+Membranes+of+stressed+Saccharomyces+cerevisiae+are+permeable+to+propidium+iodide.">Red but not dead? Membranes of stressed Saccharomyces cerevisiae are permeable to propidium iodide.</a> Environ. Microbiol. 13, 163-171 (2011).</li><li>Rodr&#237;guez-Casuriaga, R., Geisinger, A., L&#243;pez-Carro, B., Porro, V., Wettstein, R., Folle, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-fast+and+optimized+method+for+the+preparation+of+rodent+testicular+cells+for+flow+cytometric+analysis.">Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.</a> Biol. Proced. Online. 11, 184-195 (2009).</li><li>Span&#242;, M., Evenson, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometric+analysis+for+reproductive+biology.">Flow cytometric analysis for reproductive biology.</a> Biol Cell. 78, 53-62 (1993).</li></ol>

The optimized method described here enables the preparation of cell suspensions from rodent testicular tissue in a very fast and reproducible way, avoiding enzyme and detergent treatment and maintaining good cell integrity and type proportions. The brevity of the procedure (the 15 min span includes testis dissection, tissue cutting, and processing), minimal handling involved, and absence of enzymatic treatments are some of the main advantages. All these would account for the good preservation of short life macromolecules...

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Medimachine system</td>
 <td>BD</td>
 <td>340587</td>
 <td></td>
 </tr>
 <tr>
 <td>Medicon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340591</td>
 <td></td>
 </tr>
 <tr>
 <td>Filcon unit (50 &mu;m)</td>
 <td>BD</td>
 <td>340603</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Gibco</td>
 <td>430-2100</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>PAA</td>
 <td>A11-151</td>
 <td></td>
 </tr>
 <tr>
 <td>NDA</td>
 <td>Chemos BmbH</td>
 <td>277081</td>
 <td></td>
 </tr>
 <tr>
 <td>Hoechst 33342</td>
 <td>Sigma-Aldrich</td>
 <td>14533</td>
 <td>Stock solution 5 mg/ml; used at a final concentration of 5 &mu;g/ml</td>
 </tr>
 <tr>
 <td>Propidium iodide</td>
 <td>Sigma-Aldrich</td>
 <td>287075</td>
 <td>Stock solution 1 mg/ml; used at a final concentration of 50 &mu;g/ml</td>
 </tr>
 </tbody>
</table>

simple and efficient technique for the preparation of testicular cell suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

An example of a well disaggregated cell suspension from rat testes prepared with the protocol described here is shown in Figure 1. 
In comparison to enzymatic treatments6,8 and to previously described mechanical disaggregation methods2, the one presented here is much faster, involves less handling, is easily reproducible (not operator-dependant), and renders scarce cell debris (especially compared to other mechanical methods) and very few multinucleates (...

Watch this Scientific Journal Video about Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions at JoVE.com

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

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18.6K Views.  Instituto de Investigaciones Biológicas Clemente Estable (IIBCE). A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Video: Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Preparation of Single-Cell Suspensions from Mouse Spleen with the gentleMACS Dissociator

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual dissociations the gentleMACS Dissociator allows one to dissociate tissue very efficiently under controlled and reproducible conditions.  The gentleMACS Dissociator can optimally dissociate mouse spleen, combining timesaving and standardization with user-safety.

This video describes how to dissociate mouse spleens using the gentleMACS Dissociator, an automated bench-top device that can mechanically disrupt tissues using special tubes to produce viable cell suspensions. Following dissociation, spleens are filtered, centrifuged, and resuspended for further applications.

This video describes a simple, time-saving technique for automated tissue dissociation using the gentleMACS Dissociator to prepare single-cell suspensions of mouse splenocytes.

Single-cell suspensions are a prerequisite for experiments in cell separation, cell analysis and cell culture.  To avoid tedious and often painful manual ...

Standardized Preparation of Single-Cell Suspensions from Mouse Lung Tissue using the gentleMACS Dissociator

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of dissociating whole organs requires specific parameters in order to obtain a high number of viable cells in a reproducible manner. The gentleMACS Dissociator optimizes this task with a simple, practical protocol. The instrument contains pre-programmed settings that are optimized for the efficient but gentle dissociation of a variety of tissue types, including mouse lungs. In this publication the use of the gentleMACS Dissociator on lung tissue derived from mice is demonstrated.

Dissociating cells from specific tissue types requires specific parameters for tissue agitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on lung tissue is explained.

The preparation of single-cell suspensions from tissues is an important prerequisite for many experiments in cellular research. The process of ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Preparation of Quality Inositol Pyrophosphates

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells are inositol pentakisphosphate (IP5) and inositol hexakisphosphate (phytic acid or IP6). IP5 and IP6 are the precursors of inositol pyrophosphate molecules that contain one or more pyrophosphate bonds1. Phosphorylation of IP6 generates diphoshoinositolpentakisphosphate (IP7 or PP-IP5) and bisdiphoshoinositoltetrakisphosphate (IP8 or (PP)2-IP4). Inositol pyrophosphates have been isolated from all eukaryotic organisms so far studied. In addition, the two distinct classes of enzymes responsible for inositol pyrophosphate synthesis are highly conserved throughout evolution2-4.
The IP6 kinases (IP6Ks) posses an enormous catalytic flexibility, converting IP5 and IP6 to PP-IP4 and IP7 respectively and subsequently, by using these products as substrates, promote the generation of more complex molecules5,6. Recently, a second class of pyrophosphate generating enzymes was identified in the form of the yeast protein VIP1 (also referred as PP-IP5K), which is able to convert IP6 to IP7 and IP87,8.
Inositol pyrophosphates regulate many disparate cellular processes such as insulin secretion9, telomere length10,11, chemotaxis12, vesicular trafficking13, phosphate homeostasis14 and HIV-1 gag release15. Two mechanisms of actions have been proposed for this class of molecules. They can affect cellular function by allosterically interacting with specific proteins like AKT16. Alternatively, the pyrophosphate group can donate a phosphate to pre-phosphorylated proteins17. The enormous potential of this research field is hampered by the absence of a commercial source of inositol pyrophosphates, which is preventing many scientists from studying these molecules and this new post-translational modification. The methods currently available to isolate inositol pyrophosphates require sophisticated chromatographic apparatus18,19. These procedures use acidic conditions that might lead to inositol pyrophosphate degradation20 and thus to poor recovery. Furthermore, the cumbersome post-column desalting procedures restrict their use to specialized laboratories.
In this study we describe an undemanding method for the generation, isolation and purification of the products of the IP6-kinase and PP-IP5-kinases reactions. This method was possible by the ability of polyacrylamide gel electrophoresis (PAGE) to resolve highly phosphorylated inositol polyphosphates20. Following IP6K1 and PP-IP5K enzymatic reactions using IP6 as the substrate, PAGE was used to separate the generated inositol pyrophosphates that were subsequently eluted in water.

Inositol pyrophosphates play an important role in human pathologies such cancer, diabetes and obesity; however, the exact mechanism of action is a matter of dispute. The lack of commercially available inositol pyrophosphates renders detailed studies problematic. Here we describe a simple protocol to produce and isolate milligrams of inositol pyrophosphates.

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH are highly regulated in yeast cells and occupy a central role in overall pH homeostasis. We describe protocols for ratiometric measurement of pH in vivo using pH-sensitive fluorophores localized to the vacuole or cytosol. Vacuolar pH is measured using BCECF, which localizes to the vacuole in yeast when introduced into cells in its acetoxymethyl ester form. Cytosolic pH is measured with a pH-sensitive GFP expressed under control of a yeast promoter, yeast pHluorin. Methods for measurement of fluorescence ratios in yeast cell suspensions in a fluorimeter are described. Through these protocols, single time point measurements of pH under different conditions or in different yeast mutants have been compared and changes in pH over time have been monitored. These methods have also been adapted to a fluorescence plate reader format for high-throughput experiments. Advantages of ratiometric pH measurements over other approaches currently in use, potential experimental problems and solutions, and prospects for future use of these techniques are also described.

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar and cytosolic pH can be measured in live yeast (S. cerevisiae) cells using ratiometric fluorescent dyes localized to specific cellular compartments. We describe procedures for measuring vacuolar pH with BCECF-AM, which localizes to the vacuole in yeast, and cytosolic pH with a cytosolic ratiometric pH-sensitive GFP (yeast pHluorin).

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Efficient Mammalian Cell Expression and Single-step Purification of Extracellular Glycoproteins for Crystallization

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time and cost efficient protocol for secreted protein expression in mammalian cells and one step purification using nickel affinity chromatography. The system is based on large scale transient transfection of mammalian cells in suspension, which greatly decreases the time to produce protein, as it eliminates steps, such as developing expression viruses or generating stable expressing cell lines. This protocol utilizes cheap transfection agents, which can be easily made by simple chemical modification, or moderately priced transfection agents, which increase yield through increased transfection efficiency and decreased cytotoxicity. Careful monitoring and maintaining of media glucose levels increases protein yield. Controlling the maturation of native glycans at the expression step increases the final yield of properly folded and functional mammalian proteins, which are ideal properties to pursue X-ray crystallography. In some cases, single step purification produces protein of sufficient purity for crystallization, which is demonstrated here as an example case.

This is a quick, cost-efficient protocol for the production of secreted, glycosylated mammalian proteins and subsequent single-step purification with sufficient yields of homogenous protein for X-ray crystallography and other biophysical studies.

Production of secreted mammalian proteins for structural and biophysical studies can be challenging, time intensive, and costly. Here described is a time ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...