Los autores desean agradecer al Dr. Philip H. Schwartz de la Comisión Nacional de Recursos Humanos de las células madre neurales en el Hospital Infantil s, Condado de Orange el Instituto de Investigación para proporcionar culturas hNSPC.

Pases células madre neurales humanas Nota: Consulte la <a href="/index/details.stp?ID=263">pases Humanos células troncales nerviosas</a> artículo sobre cómo separar y volver a suspender las células. https://www.jove.com/index/Details.stp?ID=263 Preparación de la suspensión celular para el recuento <ol><li> Lugar 25 l de 0,4% de la solución azul de tripano y 20 l de los medios de comunicación celular en un tubo eppendorf. </li><li> Resuspender las células para ser contadas golpeando suavemente el tubo que contiene las células en un volumen conocido de los medios de comunicación para crear una suspensión homogénea. Lugar 5 l de células en el tubo eppendorf de la etapa anterior. Este paso se diluye la concentración de células en un factor de 10. </li><li> Coloque la cubierta de vidrio en el hemocitómetro, y la carga de 11-12 l de la suspensión celular. <img src="/files/ftp_upload/old/262_2008_7_18_4/262_Figure-01.png" alt="262_Figure-01" width="511" height="139" /> </li></ol> El recuento de células en el hemocitómetro <ol><li> Observar toda la red del hemocitómetro en un microscopio de contraste de fase. Se centran en un cuadrante de la red (como el de rojo). <img src="/files/ftp_upload/old/262_2008_7_18_5/262_Figure-02.png" alt="262_Figure-02" width="492" height="278" /> </li><li> Células muertas o moribundas que aparecen de color azul debido a su membrana ha sido dañada y no se puede excluir el colorante azul de tripano. Las células viables no aparecen de color azul y estará rodeada por un &quot;halo&quot; de luz en contraste de fase (se &quot;fase brillante&quot;). </li><li> Cuente el número de células viables en un cuadrante (zona 1 mm 2). También se puede determinar la viabilidad celular mediante la división del número de células viables por el número total de células. Recuento de células en 3-4 cuadrantes al azar y determinar el número promedio de células por cuadrante. Dado que el volumen de un cuadrante se sabe que es de 10 ml-4 o 0,1 l, la concentración de células se puede determinar <img src="/files/ftp_upload/old/262_2008_7_18_6/262_Figure-03.png" alt="262_Figure-03" width="741" height="165" /> </li><li> Puede usar los siguientes métodos abreviados para determinar las células # / l: <img src="/files/ftp_upload/old/262_2008_7_18_7/262_Figure-04.png" alt="262_Figure-04" width="368" height="69" /> Número total de células en el tubo = # células / X l l en el tubo </li></ol>

<ol>
	<li>Marchenko, S., Flanagan, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Passaging+Human+Neural+Stem+Cells.">Passaging Human Neural Stem Cells.</a> Journal of Visualized Experiments. 7, (2007).</li><li>Caprette, D. a. v. i. d. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemacytometer.">Using a Hemacytometer.</a> BioEd Online. , (2008).</li><li>Caprette, D. a. v. i. d. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Counting+Chamber+(Hemacytometer).">Counting Chamber (Hemacytometer).</a> BioEd Online. , (2008).</li></ol>

Este protocolo describe de una manera sencilla y rápida para determinar la concentración de células para una variedad de experimentos con las células. Utilizando el método abreviado descritas en 3D, una forma rápida y precisa de determinar la concentración de células y el número total de células en un volumen determinado.

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counting human neural stem cells

El conocimiento del número exacto de células viables en un determinado volumen de una suspensión celular que se requiere para las manipulaciones rutinarias de cultivo de tejidos, como las células de revestimiento de inmunocitoquímica o para transfecciones celulares. Este protocolo describe un método sencillo y rápido para diferenciar entre células vivas y muertas y la cuantificación de la concentración celular y el número total de células con un hemocitómetro. Este procedimiento requiere en primer lugar separar las células de la superficie de crecimiento y resuspensión de ellos en los medios de comunicación. A continuación, las células se diluyen en una solución de azul tripán (idealmente a una concentración que dará a 20-50 células por cuadrante) y se coloca en el hemocitómetro. Por último, el promedio de los conteos de células viables en varios cuadrantes seleccionados al azar, dividiendo el promedio por el volumen de un cuadrante de 1 mm 2 (0,1 l) y multiplicar por el factor de dilución da el número de células por l. Al multiplicar esta concentración de células por el volumen total en l da el número total de células. Este protocolo describe contar humana madre neurales / células precursoras (hNSPCs), pero también se puede utilizar para muchos tipos de células.

Watch this Scientific Journal Video about Counting Human Neural Stem Cells at JoVE.com

Counting Human Neural Stem Cells

El conocimiento del número exacto de células viables se requiere para muchos manipulaciones de cultivo de tejidos. Este protocolo describe cómo diferenciar entre células vivas y muertas y cuantificar las células usando un hemocitómetro. A pesar de que describe contar humana madre neurales / células precursoras (hNSPCs), que puede ser utilizado para otros tipos celulares.

Conteo de células madre neurales humanas

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Research

JoVE Journal

Biology

15.5K vistas.  University of California, Irvine (UCI). El conocimiento del número exacto de células viables se requiere para muchos manipulaciones de cultivo de tejidos. Este protocolo describe cómo diferenciar entre células vivas y muertas y cuantificar las células usando un hemocitómetro. A pesar de que describe contar humana madre neurales / células precursoras (hNSPCs), que puede ser utilizado para otros tipos celulares.

Video: Counting Human Neural Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagación de la madre embrionarias humanas (ES) Células

Investigación

Biología

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

Transfección de células madre neurales humanas con el Nucleofector Amaxa

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Passaging Human Neural Stem Cells

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

Pases células madre neurales humanas

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

Inmunocitoquímica: células madre neurales humanas

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivación de células estaminales embrionarias humanas por Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Counting and Determining the Viability of Cultured Cells

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A hemacytometer is a thick glass slide with a central area designed as a counting chamber. Cell suspension is applied to a defined area and counted so cell density can be calculated. 

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. In this video, we demonstrate how cells are counted using a hemacytometer.

Contar y determinar la viabilidad de las células cultivadas

Determining the number of cells in culture is important in standardization of culture conditions and in performing accurate quantitation experiments. A ...

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Ratón epidérmico de la cresta neural de células madre (EPI-NCSC) Culturas

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Inmunoprecipitación de la cromatina de células estaminales embrionarias humanas

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

Generación de humanos CD40 activado por células B

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Congelación y descongelación células estaminales embrionarias humanas

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...