Nous sommes reconnaissants au laboratoire Martin pour des discussions utiles. Cette publication est soutenue par une subvention de l&#39;Institut canadien de recherche en santé (MOP-89783). AM est soutenu par une recherche du Canada Chaire de niveau II Award.

Etape I: Isolement de cellules B spléniques via enrichissement magnétique <ol><li> Souris expérimentales doivent être âgés de 8-12 semaines pour assurer la maturation complète du système immunitaire. </li><li> Euthanasier la souris par dislocation cervicale et tremper dans de l&#39;éthanol à 70%. Enlever chirurgicalement la rate en faisant une incision à travers la peau et le muscle de l&#39;hypochondre gauche de l&#39;abdomen et le couper en trois gros morceaux dans du tampon phosphate salin (PBS). S&#39;assurer que tous les outils et les réactifs sont stériles. </li><li> Utilisation de l&#39;extrémité en caoutchouc d&#39;un plongeur 1 ml seringue, délicatement écrasez la rate dans une passoire 70 um de cellules dans le PBS. Veillez à utiliser un mouvement de poussée plutôt que d&#39;une motion broyage broyage peut conduire à une rupture de plus (c&#39;est à dire la prolifération) des cellules. </li><li> Isoler les cellules à pas plus de 350 g pendant 5 minutes et resuspendre le culot dans du PBS. Compter les cellules resuspendues en utilisant un hématimètre. </li><li> Préparer une suspension de 1x10 8 cellules / ml dans du PBS avec du sérum de rat normal de 5% dans un tube de polystyrène stériles de 5 ml avec un capuchon (volume maximal de 2 ml par tube). </li><li> Ajouter 50 uL de EasySep Souris choix de cocktails négatif enrichissement cellules B pour chaque ml de cellules. Pipeter le mélange, le plafond du tube, et placer au réfrigérateur pendant 15 minutes. </li><li> Ajouter 100 uL Cocktail EasySep sélection biotine pour chaque ml de cellules. Pipeter le mélange, le plafond du tube, et placer au réfrigérateur pendant 15 minutes. </li><li> Ajouter 100 ul de EasySep nanoparticules magnétiques (s&#39;assurer nanoparticules sont bien mélangés et la solution est homogène) pour chaque ml de cellules. Pipeter le mélange, le plafond du tube, et le placer dans le réfrigérateur pendant 5 minutes. </li><li> Haut de la solution à 2,5 ml avec du PBS et placer dans un aimant EasySep pendant 5 minutes. Inverser l&#39;aimant et le tube et verser rapidement dans un tube en polystyrène de nouvelles. Ne pas secouer le tube comme les cellules négativement sélectionnés seront magnétique lié aux parois du tube. </li><li> Pour augmenter encore la pureté de la suspension de cellules, le tube peut être réinséré dans l&#39;aimant pour 5 minutes supplémentaires et versé dans un nouveau tube. Cela peut réduire la récupération des cellules en général. </li><li> La pureté des cellules B peut être évaluée par cytométrie de flux des marqueurs de surface des cellules B. Suite à l&#39;enrichissement, nous avons constamment voir une pureté de&gt; 95% B220 cellules positives. </li></ol> Etape II: coloration des cellules CFSE <ol><li> Resuspendre les cellules chaudes isolées dans du PBS additionné de sérum bovin 0,1% d&#39;albumine à une concentration finale de 1 x 10 6 cellules par ml. </li><li> Préparer une solution stock de 5mm de CFSE (dilué dans du diméthylsulfoxyde stérile) et ajouter 2μL pour chaque ml de cellules dans le tube. La concentration finale de 10 uM est optimal pour la coloration des cellules B primaires. </li><li> Incuber à 37 ° C pendant 10 minutes dans l&#39;obscurité (à noter: CFSE est sensible à la lumière et doit être conservé dans l&#39;obscurité quand c&#39;est possible). </li><li> Étancher la tache en ajoutant un volume égal de sérum de veau bovin à vos cellules et incubation sur la glace pendant 5 minutes. </li><li> Laver les cellules en complétant les tubes avec milieu de culture (voir l&#39;étape III pour la recette) et centrifugation à 350 g. Notez qu&#39;au moins cinq fois l&#39;équivalent du volume des médias doivent être ajoutés pour neutraliser la viscosité du sérum de veau bovin et pour les cellules à produire une boulette. </li><li> Laver les cellules deux fois plus en milieu de culture. Les cellules sont maintenant prêts pour la stimulation. </li></ol> Etape III: stimulation cellulaire par le LPS <ol><li> Préparez votre in vitro, B milieu de culture cellulaire en complétant RPMI 1640 avec du sérum de veau foetal à 10% et 50 uM β-mercaptoéthanol /. </li><li> Préparez votre support de stimulation par l&#39;ajout de LPS à une concentration finale de 50μg/mL. Aliquoter 125 ul du milieu de stimulation dans chaque puits d&#39;un fond plat de 96 puits plaque de culture tissulaire. </li><li> Préparez vos cellules B CFSE teinté en milieu de culture sans LPS à une concentration finale de 3,2 x 10 6 cellules / ml. Ajouter 125 ul de la suspension cellulaire dans les puits contenant votre support de stimulation et mélanger délicatement à la pipette. </li><li> Chaque puits contient maintenant 4 x 10 5 cellules à une concentration finale de LPS de 25 pg / mL. Cela fournit un niveau optimal de la prolifération cellulaire et la commutation de classe, bien que des modifications à ces valeurs peuvent être faites comme vous le souhaitez. </li><li> Incuber les cellules à 37 ° C et 5% de CO 2 pendant 72 à 96 heures. Après 48 heures, des grappes de grandes cellules B proliférantes sera clairement visible sous un microscope. </li></ol> Etape IV: cytométrie de flux de commutation de classe <ol><li> Lorsque vous êtes prêt à regarder le changement de classe, retirez vos cellules de leurs conditions de culture et de les laver deux fois dans 1 ml de tampon de coloration (PBS avec 2% de sérum bovin de veau). </li><li> Pour prévenir les taches d&#39;anticorps non spécifiques de vos cellules, un culot cellulaire en les faisant tourner vers le bas à 350 g et incubateursTE eux dans 100 pi de tampon avec coloration du sérum de souris normales et 5% des 1 pg FcBlock per106 cellules pendant 15 minutes sur la glace. Bloquant la cellule doit être effectué sur la glace. </li><li> Sans se laver les cellules, ajouter 1 pg par 10 6 cellules d&#39;une souris fluorescente taggés IgG3 anti-anticorps et permettre aux cellules de coloration pendant 30 minutes sur la glace. </li><li> Laver les cellules deux fois par centrifugation et resuspendre dans 200-300 ul de tampon de coloration. Les cellules sont maintenant prêts pour l&#39;évaluation par cytométrie en flux. </li><li> CFSE est parfaitement excité à 492 nm (par un laser à l&#39;argon-ion) et émet à 517 nm. Le spectre d&#39;excitation et d&#39;émission de l&#39;anticorps IgG3 est dépendante de ses colorant fluorescent conjugué. </li></ol> Les résultats représentatifs Après enrichissement magnétique, la suspension cellulaire doit ressembler à une population homogène de petites cellules indiscernables circulaire (figure 1A). Après 48-72 heures de stimulation, des groupes isolés de cellules proliférantes élargie sera clairement visible (figure 1B). Une grande proportion des cellules non-proliférantes apparaîtra petites et granuleuses comme ils subissent l&#39;apoptose. La RSE peut normalement être détectée au plus haut niveau entre 72 et 96 heures après la stimulation avant la plupart des cellules commencent à mourir 7. A ce stade, les cellules doivent avoir subi un certain nombre de divisions cellulaires avec les cellules tension apparaissant dans la population cellule fille plus tard. Une parcelle de flux représentant cytométrie de dilution CFSE et d&#39;expression de surface de l&#39;IgG3 après 96 heures de stimulation par le LPS peut être vu dans la figure 2. Tableau 1. Isotype commutation cocktails. Remarque: Les valeurs de concentration de stimulants sont que des recommandations. Titrages peut être nécessaire. <table border="1"><tbody><tr><td> Isotype désiré </td><td> Cocktail de stimulation </td></tr><tr><td> IgG1and IgE </td><td> LPS (25μg/mL) et de l&#39;IL-4 (10 ng / ml) </td></tr><tr><td> IgG1and IgE </td><td> Anti-CD40 (10μg/mL) et de l&#39;IL-4 (10 ng / ml) </td></tr><tr><td> IgG2a </td><td> LPS (25μg/mL) et l&#39;IFN-γ (10 ng / ml) </td></tr><tr><td> IgG2b et IgG3 </td><td> LPS (25μg/mL) </td></tr><tr><td> IgA </td><td> LPS (25μg/mL), le TGF-β (2 ng / mL) et IL-5 (1,5 ng / mL) </td></tr></tbody></table><img src="/files/ftp_upload/2130/2130fig1.jpg" alt="Figure 1" /> Figure 1. Isolement et stimulation par le LPS des cellules B spléniques. (A) magnétiquement enrichi cellules B spléniques, avant stimulation par le LPS. (B) 48 heures après la stimulation avec 25 pg / mL de LPS. Les cellules en prolifération sont considérés comme des clusters dans un fond de cellules apoptotiques et de dynamitage. <img src="/files/ftp_upload/2130/2130fig2.jpg" alt="Figure 2" /> Figure 2. Prolifération et la commutation isotypique après 96 heures. (A) dilution CFSE pour les cellules stimulées par LPS après 96 heures de culture. Chaque pic indépendante représente une dilution de la fluorescence CFSE correspondant à une division cellulaire indépendant. (B) l&#39;expression IgG3 montrant l&#39;émergence d&#39;une population IgG3 positif après plusieurs cycles de division cellulaire.

<ol>
	<li>Hodgkin, P. D., Lee, J. H., Lyons, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B+cell+differentiation+and+isotype+switching+is+related+to+division+cycle+number.">B cell differentiation and isotype switching is related to division cycle number.</a> J Exp Med. 184, 277-281 (1996).</li><li>McCall, M. N., Hodgkin, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+recombination+and+germ-line+transcription+are+division-regulated+events+in+B+lymphocytes.">Switch recombination and germ-line transcription are division-regulated events in B lymphocytes.</a> Biochim Biophys Acta. 1447, 43-50 (1999).</li><li>Manis, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=53BP1+links+DNA+damage-response+pathways+to+immunoglobulin+heavy+chain+class-switch+recombination.">53BP1 links DNA damage-response pathways to immunoglobulin heavy chain class-switch recombination.</a> Nat Immunol. 5, 481-487 (2004).</li><li>de Yebenes, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-181b+negatively+regulates+activation-induced+cytidine+deaminase+in+B+cells.">miR-181b negatively regulates activation-induced cytidine deaminase in B cells.</a> J Exp Med. 205, 2199-2206 (2008).</li><li>McBride, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+class+switch+recombination+and+somatic+mutation+by+AID+phosphorylation.">Regulation of class switch recombination and somatic mutation by AID phosphorylation.</a> J Exp Med. 205, 2585-2594 (2008).</li><li>Ramachandran, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNF8/RNF168+ubiquitin+ligase+cascade+facilitates+class+switch+recombination.">The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination.</a> Proc Natl Acad Sci U S A. 107, 809-8014 (2010).</li><li>Zaheen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AID+constrains+germinal+center+size+by+rendering+B+cells+susceptible+to+apoptosis.">AID constrains germinal center size by rendering B cells susceptible to apoptosis.</a> Blood. 114, 547-554 (2009).</li><li>Chaudhuri, J., Alt, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Class-switch+recombination:+interplay+of+transcription,+DNA+deamination+and+DNA+repair.">Class-switch recombination: interplay of transcription, DNA deamination and DNA repair.</a> Nat Rev Immunol. 4, 541-552 (2004).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimuli+that+enhance+IgA+class+switching+increase+histone+3+acetylation+at+S+alpha,+but+poorly+stimulate+sequential+switching+from+IgG2b.">Stimuli that enhance IgA class switching increase histone 3 acetylation at S alpha, but poorly stimulate sequential switching from IgG2b.</a> Eur J Immunol. 37, 240-251 (2007).</li><li>Kaminski, D. A., Stavnezer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+class+switching+differs+among+SJL,+C57BL/6+and+129+mice.">Antibody class switching differs among SJL, C57BL/6 and 129 mice.</a> Int Immunol. 19, 545-556 (2007).</li></ol>

Le protocole décrit ci-dessus donne un dosage standard pour analyser l&#39;expression AID et la fonction lors de la commutation de classe du primaire des cellules B murines. Ce protocole utilise LPS bactérien pour induire le passage d&#39;IgM IgG3, même si des modifications aux médias stimulation peut être faite pour induire le passage à d&#39;autres isotypes (résumées dans le Tableau 1 8, 9). Nous avons noté que, pour des raisons encore inconnues, du sérum de veau fœta...

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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Phosphate Buffered Saline</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>14190</td>
<td>&nbsp;</td>
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<td>EasySep Mouse B Cell Enrichment Kit</td>
<td>&nbsp;</td>
<td>Stemcell Technologies</td>
<td>19754</td>
<td>&nbsp;</td>
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<td>Polystyrene tubes</td>
<td>&nbsp;</td>
<td>Becton Dickinson Falcon</td>
<td>352058</td>
<td>&nbsp;</td>
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<td>Bovine Serum Albumin</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>A7030</td>
<td>&nbsp;</td>
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<td>CellTrace CFSE Cell Proliferation Kit</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C34554</td>
<td>&nbsp;</td>
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<td>Bovine Calf Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30072.03</td>
<td>&nbsp;</td>
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<td>RPMI 1640 Culture Medium</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11875</td>
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<td>Lipopolysaccharides from Escherichia coli</td>
<td>&nbsp;</td>
<td>Sigma Aldrich</td>
<td>L6529</td>
<td>&nbsp;</td>
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<td>&beta;-mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>21985</td>
<td>&nbsp;</td>
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<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>HyClone</td>
<td>SH30396.03</td>
<td>&nbsp;</td>
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<td>Normal Mouse Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>011-000</td>
<td>&nbsp;</td>
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<td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553141</td>
<td>&nbsp;</td>
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<td>Rat Anti-Mouse IgG3</td>
<td>&nbsp;</td>
<td>Becton Dickinson Pharmingen</td>
<td>553401</td>
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induction and assessment of class switch recombination in purified murine b cells

L&#39;immunité humorale est la branche du système immunitaire entretenu par les cellules B et médiée par la sécrétion d&#39;anticorps. Lors de l&#39;activation des lymphocytes B, le locus d&#39;immunoglobuline subit une série de modifications génétiques pour modifier la capacité de liaison et de la fonction effectrice des anticorps sécrétés. Ce processus est mis en évidence par un événement de recombinaison génomique connue comme la commutation isotypique (RSE) dans lequel l&#39;isotype des anticorps IgM par défaut est substitué à l&#39;un des IgG, IgA ou IgE. Chaque isotype possède des fonctions effectrices distinctes rendant ainsi la RSE crucial pour le maintien de l&#39;immunité. Diversification de l&#39;locus d&#39;immunoglobuline est médiée par l&#39;enzyme cytidine désaminase induite par activation (AID). Une vidéo décrivant schématiquement ce processus en détail est disponible en ligne (http://video.med.utoronto.ca/videoprojects/immunology/aam.html). L&#39;activité de l&#39;AID et la voie de la RSE sont fréquemment étudiés dans l&#39;évaluation de la fonction des cellules B et l&#39;immunité humorale chez la souris. Le protocole décrit dans le présent rapport présente une méthode d&#39;isolement des cellules B de la rate de souris et une stimulation ultérieure avec lipopolysaccharide (LPS) à induire le passage à la classe IgG3 (pour les autres isotypes d&#39;anticorps voir tableau 1). En outre, les cellules fluorescentes coloration colorants carboxyfluorescéine succinimidyl ester (CFSE) est utilisé pour surveiller la division cellulaire des cellules stimulées, un processus crucial pour la commutation isotypique 1, 2. Le règlement de l&#39;AID et le mécanisme par lequel la RSE se sont pas encore claires et donc passer des tests in vitro de classe fournissent une méthode fiable pour les tests de ces processus dans les divers modèles de souris. Ces tests ont déjà été utilisés dans le contexte de déficience du gène en utilisant des souris knockout 3. Par ailleurs, in vitro, de commutation de cellules B peut être précédée par transduction virale pour moduler l&#39;expression des gènes par ARN ou knockdown l&#39;expression du transgène 4-6. Les données de ces types d&#39;expériences ont influencé notre compréhension des activités d&#39;aide, la résolution de la réaction de la RSE, et l&#39;immunité à médiation d&#39;anticorps chez la souris.

Watch this Scientific Journal Video about Induction and Assessment of Class Switch Recombination in Purified Murine B Cells at JoVE.com

Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Après l&#39;exposition des antigènes, des sous-populations de cellules B activées subissent un processus connu sous le nom commutation isotypique (CSR) pour produire des isotypes d&#39;anticorps avec les différentes fonctions effectrices. Le protocole décrit dans le présent rapport explique comment la RSE peut être induite et analysé<em&gt; In vitro</em&gt; Aux fins d&#39;étudier la fonction des cellules B.

Induction et d&#39;évaluation de la recombinaison commutateur de classe dans purifiée cellules B murines

Humoral immunity is the branch of the immune system maintained by B cells and mediated through the secretion of antibodies. Upon B cell activation, the ...

induction-assessment-class-switch-recombination-purified-murine-b

Research

JoVE Journal

Immunology and Infection

18.0K vues.  University of Toronto. Après l'exposition des antigènes, des sous-populations de cellules B activées subissent un processus connu sous le nom commutation isotypique (CSR) pour produire des isotypes d'anticorps avec les différentes fonctions effectrices. Le protocole décrit dans le présent rapport explique comment la RSE peut être induite et analysé In vitro Aux fins d'étudier la fonction des cellules B.

Vidéo: Induction and Assessment of Class Switch Recombination in Purified Murine B Cells

Murine Model of CD40-activation of B cells

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand (CD40L), human B cells can be expanded without difficulties from small amounts of peripheral blood within 14 days to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients1-4. CD40-B cells express important lymph node homing molecules and can attract T cells in vitro5. Furthermore they efficiently take up, process and present antigens to T cells6,7. CD40-B cells were shown to not only prime na&iacute;ve, but also expand memory T cells8,9. Therefore CD40-activated B cells (CD40-B cells) have been studied as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cell-based immunotherapy1,5,10. In order to further study whether CD40-B cells induce effective T cell responses in vivo and to study the underlying mechanism we established a cell culture system for the generation of murine CD40-activated B cells. Using splenocytes or purified B cells from C57BL/6 mice for CD40-activation, optimal conditions were identified as follows: Starting from splenocytes of C57BL/6 mice (haplotype H-2b) lymphocytes are purified by density gradient centrifugation and co-cultured with HeLa cells expressing recombinant murine CD40 ligand (tmuCD40L HeLa)11. Cells are recultured every 3-4 days and key components such as CD40L, interleukin-4, -Mercaptoethanol and cyclosporin A are replenished. In this protocol we demonstrate how to obtain fully activated murine CD40-B cells (mCD40B) with similar APC-phenotype to human CD40-B cells (Fig 1a,b). CD40-stimulation leads to a rapid outgrowth and expansion of highly pure (&gt;90%) CD19+ B cells within 14 days of cell culture (Fig 1c,d). To avoid contamination with non-transfected cells, expression of the murine CD40 ligand on the transfectants has to be controlled regularly (Fig 2). Murine CD40-activated B cells can be used to study B-cell activation and differentiation as well as to investigate their potential to function as APC in vitro and in vivo. Moreover, they represent a promising tool for establishing therapeutic or preventive vaccination against tumors and will help to answer questions regarding safety and immunogenicity of this approach12.

In this video, we demonstrate the procedure of CD40-activation and expansion of murine B cells from splenocytes of C57BL/6 mice, which can be used as a model antigen-presenting cell (APC) to study induction of immunity.

Modèle murin de CD40-activation des cellules B

Research on B cells has shown that CD40 activation improves their antigen presentation capacity. When stimulated with interleukin-4 and CD40 ligand ...

Recherche

Immunologie et infection

Identifying DNA Mutations in Purified Hematopoietic Stem/Progenitor Cells

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem cells are responsible for ensuring tissue homeostasis and repair throughout life, it is reasonable to hypothesize that the stem cell population is critical for preserving genomic integrity of tissues. Therefore, significant interest has arisen in assessing the impact of endogenous and environmental factors on genomic integrity in stem cells and their progeny, aiming to understand the etiology of stem-cell based diseases.
LacI transgenic mice carry a recoverable &#955;&#160;phage vector encoding the LacI reporter system, in which the LacI gene serves as the mutation reporter. The result of a mutated LacI gene is the production of &#946;-galactosidase that cleaves a chromogenic substrate, turning it blue. The LacI reporter system is carried in all cells, including stem/progenitor cells and can easily be recovered and used to subsequently infect E. coli. After incubating infected E. coli on agarose that contains the correct substrate, plaques can be scored; blue plaques indicate a mutant LacI gene, while clear plaques harbor wild-type. The frequency of blue (among clear) plaques indicates the mutant frequency in the original cell population the DNA was extracted from. Sequencing the mutant LacI gene will show the location of the mutations in the gene and the type of mutation.
The LacI transgenic mouse model is well-established as an in vivo mutagenesis assay. Moreover, the mice and the reagents for the assay are commercially available. Here we describe in detail how this model can be adapted to measure the frequency of spontaneously occurring DNA mutants in stem cell-enriched Lin-IL7R-Sca-1+cKit++(LSK) cells and other subpopulations of the hematopoietic system.

Here we describe an in vivo mutagenesis assay for small numbers of purified hematopoietic cells using the LacI transgenic mouse model.&#160;The LacI gene can be isolated to determine the frequency, location, and type of DNA mutants&#160;spontaneously arisen or after exposure to genotoxins.

Identifier ADN mutations dans purifié hématopoïétiques cellules souches / progénitrices

In recent years, it has become apparent that genomic instability is tightly related to many developmental disorders, cancers, and aging. Given that stem ...

Detection of True IgE-expressing Mouse B Lineage Cells

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH isotypes, such as IgA, IgE and IgG. Measurement of IgH CSR in vitro is a key method for the study of a number of biologic processes ranging from DNA recombination and repair to aspects of molecular and cellular immunology. In vitro CSR assay involves the flow cytometric measurement surface Ig expression on activated B cells. While measurement of IgA and IgG subclasses is straightforward, measurement of IgE by this method is problematic due to soluble IgE binding to Fc&#949;RII/CD23 expressed on the surface of activated B cells. Here we describe a unique procedure for accurate measurement of IgE-producing mouse B cells that have undergone CSR in culture. The method is based on trypsin-mediated cleavage of IgE-CD23 complexes on cell surfaces, allowing for detection of IgE-producing B lineage cells by cytoplasmic staining. This procedure offers a convenient solution for flow cytometric analysis of CSR to IgE.

In vitro analysis of class switch recombination in mice is challenging due to cytophilic IgE molecules bound to Fc receptors on the surface of B cells. We describe a method for IgE detection using trypsin-mediated cleavage of surface-bound IgE prior to fixation and permeabilization for cytoplasmic fluorescence staining.

Détection de True IgE exprimant Souris B cellules de la lignée

B lymphocyte immunoglobulin heavy chain (IgH) class switch recombination (CSR) is a process wherein initially expressed IgM switches to other IgH ...

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of na&#239;ve effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Induction of Murine Intestinal Inflammation by Adoptive Transfer of Effector CD4+CD45RBhigh T Cells into Immunodeficient Mice

Here, we present a protocol to induce colonic inflammation in mice by adoptive transfer of syngeneic CD4+CD45RBhigh T cells into T and B cell deficient recipients. Clinical and histopathological features mimic human inflammatory bowel diseases. This method allows the study of the initiation of colonic inflammation and progression of disease.

Induction de murin inflammation intestinale par transfert adoptif de effecteur CD4<sup&gt; +</sup&gt; CD45RB<sup&gt; Haute</sup&gt; T cellules dans des souris immunodéficientes

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

Évaluation de la propriétés immunomodulatrices des mésenchymateuses humaines cellules souches (MSC)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Un modèle murin du Groupe B<em&gt; Streptococcus</em&gt; Vaginal Colonisation

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Genome-wide Analysis of HDAC Inhibitor-mediated Modulation of microRNAs and mRNAs in B Cells Induced to Undergo Class-switch DNA Recombination and Plasma Cell Differentiation

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response. Here, we demonstrate a protocol for inducing B cells to undergo CSR and plasma cell differentiation, treating these B cells with histone deacetylase (HDAC) inhibitors (HDIs), and analyzing mRNA and microRNA expression. In this protocol, we directly analyze complementary DNA (cDNA) sequences using next-generation mRNA sequencing (mRNA-seq) and miRNA-seq technologies, mapping of the sequencing reads to the genome, and quantitative reverse transcription (qRT)-PCR. With these approaches, we have defined that, in B cells induced to undergo CSR and plasma cell differentiation, HDI, an epigenetic regulator, selectively modulates miRNA and mRNA expression and alters CSR and plasma cell differentiation.

Epigenetic factors can interact with genetic programs to modulate gene expression and regulate B cell function. By combining in vitro B-cell stimulation, qRT-PCR, and high-throughput microRNA-sequence and mRNA-sequence approaches, we can analyze the epigenetic modulation of miRNA and gene expression in B cells.

Modulation de génome induite par l’analyse des HDAC inhibiteur des microARN et ARNm dans B cellules amenées à soumises classe-commutateur de recombinaison de l’ADN et la différenciation des cellules de Plasma

Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

In Vitro et In Vivo l’évaluation d’activité Suppressive T, B et les cellules myéloïdes et des réponses humorales de receveurs de transplantation

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Analysis of Shear Flow-induced Migration of Murine Marginal Zone B Cells In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, MZBs must migrate up the shear force of blood flow. We present here a method for analyzing this flow-induced MZB migration in vitro. First, MZBs are isolated from the mouse spleen. Second, MZBs are settled on integrin ligands in flow chamber slides, exposed to shear flow, and imaged under a microscope while migrating. Third, images of the migrating MZBs are processed using the MTrack2 automatic cell tracking plugin for ImageJ, and the resulting cell tracks are quantified using the Ibidi chemotaxis tool. The migration data reveal how fast the cells move, how often they change direction, whether the shear flow vector affects their migration direction, and which integrin ligands are involved. Although we use MZBs, the method can easily be adapted for analyzing migration of any leukocyte that responds to the force of shear flow.

Marginal zone B cells (MZBs) respond to the force of shear flow by re-orienting their migration path up the flow. This protocol shows how to record and analyze the migration using a fluidics unit, pump, microscope imaging system, and free software.

Analyse de cisaillement induite par le flux de Migration des cellules B Zone marginale murin In Vitro

Marginal zone B cells (MZBs) are a population of B cells that reside in the mouse splenic marginal zones that envelop follicles. To reach the follicles, ...

Studying Organelle Dynamics in B Cells During Immune Synapse Formation

Recognition of surface-tethered antigens&#160;by the B cell receptor (BCR)&#160;triggers the formation of an immune synapse (IS), where both signaling&#160;and antigen uptake are coordinated. IS formation involves dynamic actin remodeling accompanied by the polarized recruitment to the synaptic membrane of the centrosome and associated intracellular organelles such as lysosomes and the Golgi apparatus. Initial stages of actin remodeling allow B cells to increase their cell surface and maximize the quantity of antigen-BCR complexes gathered at the synapse. Under certain conditions, when B cells recognize antigens associated to rigid surfaces, this process is coupled to the local recruitment and secretion of lysosomes, which can facilitate antigen extraction. Uptaken antigens&#160;are internalized into specialized endo-lysosome compartments for processing into peptides, which are loaded onto major histocompatibility complex II (MHC-II) molecules for further presentation to T helper cells. Therefore, studying organelle dynamics associated with the formation of an IS is crucial to understanding how B cells are activated. In the present article we will discuss both imaging and a biochemical technique used to study changes in intracellular organelle positioning and cytoskeleton rearrangements that are associated with the formation of an IS in B cells.

Herein we describe two approaches to characterize cell polarization events in B lymphocytes during the formation of an IS. The first, involves quantification of organelle recruitment and cytoskeleton rearrangements at the synaptic membrane. The second is a biochemical approach, to characterize changes in composition of the centrosome, which undergoes polarization to the immune synapse.