In order to examine whether or not soluble antigen induces RAG ex

In order to examine whether soluble antigen induces RAG expression in antigen activated B cells, mg of soluble BSA or BSAwas injected i.v. into KLHeimmunized mice on days and after the key challenge. Adoptive transfer BALB c or Bcl Tg mice were immunized with DWEYS MAP as described above. mMT mice had been immunized with DEWYS MAP two weeks before adoptive transfer. Spleens from BALB c or Bcl Ig mice have been harvested on day following immunization. B cells were purified by using the mouse B cell isolation kit . In order to take out plasma cells, anti mouse CD biotin was additional to the antibody cocktail. of purified splenic B cells cells mouse had been injected i.v. into peptide primed mMT mice. Recipient mice had been boosted the next day following cell transfer. Tetramer generation DWEYSVWLSN streptavidin allophycocyanin tetramers, andDWEYSVWLSN streptavidin AlexaFluor tetramers were produced as previously described . Biotinylated peptide was synthesized by AnaSpec. Streptavidin APC and streptavidin AlexaFluor was purchased from Invitrogen . Reagents and flow cytometry Mice were sacrificed and spleens had been harvested around the specified date and placed in cold Hanks Balanced Salt Choice supplemented with .
fetal calf serum . Single cell suspensions have been produced by grinding spleens on the mm cell strainer. The next anti mouse antibodies had been implemented for flow cytometry examination: PerCP anti B , APC DWEYS tetramer was utilized to detect antigenbinding B cells. Diamidino phenylindole was additional before flow cytometry to exclude dead cells. Erythrocytes were lysed in . M NHCl, pH Cells had been stained in Trametinib cost selleckchem HBSS FCS at C for min. Data have been acquired by utilizing LSRII movement cytometry and analyzed through the use of FlowJo software package . ELISA ELISA plates have been coated with mg ml DWEYS MAP or mg ml sonicated, filtered calf thymus DNA . Plates were blocked with FCS. Serum antibody was detected following washing in PBS Tween with mg ml anti mouse IgG AP in PBS BSA. Plates were washed and binding was measured by addition of mg ml PNPP and go through at nm on the Titertek Multiskan Plus. Cell sorting To type the tetramer binding populations, splenocytes had been ready from to mice at day soon after immunization.
T cells, monocytes and dendritic cells have been depleted as previously reported . Staining was performed as described over. At once after sorting, cells have been resuspended in Trizol and frozen at C until finally RNA isolation. Sorting was performed on a FACSAria Flow Cytometer . Spleens had been removed on day or after the initial immunization and frozen in Tissue Tek OCT Compound by immersion in the methylbutane bath on dry ice. Sections were cut using a Leica CM cryostat. Before staining, Roscovitine selleck sections had been warmed to room temperature and rehydrated in PBS followed by blocking for min with FCS. Staining was carried out in FCS plus . Triton X for min at space temperature.

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