Name: Murine Model of CD40-activation of B cells (Video) | JoVE Journal
Uploaded: 2010-03-05T17:30:09.000+00:00
Duration: 12 min 24 s
Description: Learn CD40-activation of murine B cells from C57BL/6 splenocytes using IL-4 stimulation and feeder cell culture. This method enables generation of antigen-presenting cells for immunology research.

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5 minutes

We kindly thank Dr. Clemens Wendtner for providing tmuCD40-Ligand-HeLa cell line.

The protocol for the generation of murine CD40-activated B cells (mCD40B) from splenocytes is divided into two parts: Part A demonstrates the preparation of murine CD40 ligand (CD40L) expressing HeLa cells (tmuCD40L HeLa), which will be used as plate-bound feeder cells. Part B describes the actual murine CD40-B culture.
A. Preparation of feeder cells (tmuCD40L HeLa)
The tmuCD40L HeLa is an adherent human epithelial cell line, which should never become completely confluent. The cells are therefore splitted twice per week. Culturing over more than 6 weeks is not recommended.
<ol>
 <li>Remove old medium from the primary culture with a sterile pipette and wash cells with 10 mL of 1x PBS. Aspirate the PBS after washing.</li>
 <li>Add 4 mL of 1x Trypsin/EDTA in a 75 cm2 flask for 10-15 minutes at 37°C. Use gentle tapping to detach the cells.</li>
 <li>Add 10 mL of wild type medium and swivel gently to stop digestion with trypsin.</li>
 <li>Transfer the cell suspension into a 50 mL tube with a sterile pipette and spin the cells down at 265 x g for 7 minutes.</li>
 <li>Remove the supernatant and resuspend the cell pellet in 10 mL of wild type medium. Count the cell number of an aliquot of the cell suspension and prepare three 50 mL tubes with the appropriate number of cells:
 <ol>
 <li>2.5 x 106 cells for subculture</li>
 <li>0.4 x 106 cells/well for irradiation used for the murine CD40-B cell culture</li>
 <li>remainder to freeze (if needed).</li>
 </ol>
 </li>
 <li>Spin the cells down at 265 x g for 7 minutes.</li>
 <li>Remove the supernatant.
 <ol>
 <li>For subculturing: resuspend 2.5 x 106 cells in 10 mL selection medium in a 75 cm2 cell culture flask (cell density 2.5 x 105 cells/mL) and incubate the cells at 37°C with 5 % CO2. Split the cells twice per week.</li>
 <li>For the murine CD40-B cell culture: You need 2.4 x 106 cells for one 6-well plate. Resuspend the tmuCD40L HeLa cells in wild type medium at a density of 0.2 x 106 cells/mL and irradiate them at 78 Gy. Plate 2 mL of the cell suspension into each well and incubate them at 37°C with 5 % CO2. Use this prepared plates for B-cell stimulation when tmuCD40L HeLa cells are adherent (at least 4 hours: check adherence using the microscope; do not wait more than 24 h to start B-cell stimulation). (Continue with B.)</li>
 </ol>
 </li>
</ol>
B. Murine CD40-B cell culture
I. Preparation of splenocytes for CD40-stimulation (day 0):
Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of murine interleukin-4, β-Mercaptoethanol and cyclosporin A to the growth medium immediately before use.
<ol>
 <li>Dissect spleen of C57BL/6 mice (haplotype H-2b) and wash it twice in 10-20 mL DMEM supplemented with Gentamycin [15μg/mL] by transferring it with a 10mL pipette tip from one tube to another. Avoid transferring contaminants (e.g. hair) of the mouse. Mince spleen by passing it through a cell strainer (100μm) and rinse strainer with another 10 mL of medium. Spin down splenocytes at 265 x g for 7 min at room temperature, resuspend them in 7mL muCD40-B cell medium, and separate lymphocytes by Ficoll density centrifugation. To do so layer cells on 5 mL Mouse-Pancoll (preheated at room temperature) in a 15 mL tube and centrifuge at 450 x g for 30 min without brake at room temperature. </li>
 <li>Draw off most of the upper layer, leaving the lymphocyte layer undisturbed at the interface. Transfer lymphocyte layer to a fresh 50 mL tube, wash once with 30mL DMEM with Gentamycin [15μg/mL] by spinning down at 265 x g for 7 min to remove other cells. Discard the supernatant and resuspend the cells in 10 mL of mCD40-B washing medium. Determine the cell number in an aliquot of the cell suspension. Determine cell number by counting an aliquot of the cell suspension.</li>
 <li>Spin down the required amount of cells at 265 x g for 7 minutes. For a 6-well plate 5 x 106 cells/well are needed, thus 30 x 106 cells per plate. </li>
 <li>Remove the supernatant and resuspend the lymphocytes at 1.25 x 106 cells/mL in mCD40-B culture medium freshly supplemented with 1 U/mL of interleukin-4 as growth factor, 100 μM β-Mercaptoethanol and 0.63 μg/mL cyclosporin A to prevent outgrowth of T-cells (Given concentrations refer to one mL culture medium!). </li>
 <li>Remove the supernatant from the 6-well plate pre-incubated with tmuCD40L HeLa cells. </li>
 <li>Gently add 4 mL of the lymphocyte suspension (1.25 x 106 cells/mL) to each well of the 6-well plate. </li>
 <li>Incubate the plate at 37°C with 5 % CO2. </li>
 <li>On day 3 reculture the cells (Continue with II.1.). </li>
</ol>
II. Subculture of mCD40B cells (day 3 and then twice a week):
<ol>
 <li>Harvest mCD40B cell cluster by vigorously pipetting up and down with a 10 mL pipette and pool them into a 50 mL tube.</li>
 <li>Spin down at 265 x g for 7 minutes and completely replace the supernatant with mCD40-B washing medium. While counting the cell amount of an aliquot, spin the cells down at 265 x g for 7 minutes. Resuspend mCD40B cells in mCD40-B culture medium at a concentration of 0.75 x 106 cells/mL.</li>
 <li>Add fresh solutions of murine interleukin-4 in a concentration of 1 U/mL, 100 μM β-Mercaptoethanol and 0.63 μg/mL cyclosporin A to the medium.</li>
 <li>Remove the supernatant from a 6-well plate pre-incubated with irradiated tmuCD40L HeLa cells.</li>
 <li>Gently add 4 mL of the mCD40B cell suspension (0.75 x 106cells/mL) to each well of the 6-well plate.</li>
 <li>Incubate plates at 37°C with 5 % CO2.</li>
 <li>Again subculture the cells every 3-4 days to end up with highly pure murine CD40-activated B cells after a total of 14 days. </li>
</ol>
C. Trouble-shooting: What if CD40-B cells do not grow?
<ol>
 <li>Have you checked for CD40 ligand expression of feeder cells?</li>
 <li>Are the plate-bound feeder cells used for the stimulation older than 24h?</li>
 <li>Is there a contamination with mycoplasma?</li>
 <li>Was the interleukin-4 solution used for supplementation freshly thawed and did it have the appropriate biological activity?</li>
 <li>Was β-Mercaptoethanol and cyclosporin A added in the correct concentration?</li>
</ol>

<img src="/files/ftp_upload/1734/1734fig1a.jpg" alt="Phenotypical FACS diagram, CD19⁺ mCD40B cell analysis, CD40-stimulation, immunophenotyping." data-altupdatedat="1788235205000" data-alt="Figure 1 a"> Figure 1a.
<img src="/files/ftp_upload/1734/1734fig1b.jpg" alt="Flow cytometry graph; lymphocyte, mCD40B clustering analysis with CD19, CD3 markers; cell aggregation." data-altupdatedat="1788235205000" data-alt="Figure 1 b"> Figure 1b.
<img src="/files/ftp_upload/1734/1734fig1d.jpg" alt="Quantification of mCD40B cell growth; graph of B and T cell culture growth over 14 days." data-altupdatedat="1788235205000" data-alt="Figure 1 d"> Figure 1d.
<img src="/files/ftp_upload/1734/1734fig2.jpg" alt="Flow cytometry diagram; SSC vs. FSC dot plot and CD154 histogram for HeLa cell gating analysis." data-altupdatedat="1788235205000" data-alt="Figure 2"> Figure 2.

<ol><li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD40-activated+human+B+cells%3A+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy%5BTitle%5D)+AND+%22J+Clin+Invest%22%5BJournal%5D)">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100 (11), 2757-2765 (1997).</li>
<li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conditional+immortalization+of+human+B+cells+by+CD40+ligation%5BTitle%5D)+AND+%22PLoS+ONE%22%5BJournal%5D)">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, e1464-e1464 (2008).</li>
<li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+generation+of+antigen-specific+cytotoxic+T+cells+using+retrovirally+transduced+CD40-activated+B+cells%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Efficient generation of antigen-specific cytotoxic T cells using retrovirally transduced CD40-activated B cells.</a> J Immunol. 169 (4), 2164-2171 (2002).</li>
<li>Liebig, T. M., Fiedler, A., Zoghi, S., Shimabukuro-Vornhagen, A., Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+human+CD40-activated+B+cells%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Generation of human CD40-activated B cells.</a> J Vis Exp. 32, (2009).</li>
<li>von Bergwelt-Baildon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis%3A+potential+as+cellular+adjuvants%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107 (7), 2786-2789 (2006).</li>
<li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63 (11), 2836-2843 (2003).</li>
<li>Ahmadi, T., Flies, A., Efebera, Y., Sherr, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD40+Ligand-activated%2C+antigen-specific+B+cells+are+comparable+to+mature+dendritic+cells+in+presenting+protein+antigens+and+major+histocompatibility+complex+class+I-+and+class+II-binding+peptides%5BTitle%5D)+AND+%22Immunology%22%5BJournal%5D)">CD40 Ligand-activated, antigen-specific B cells are comparable to mature dendritic cells in presenting protein antigens and major histocompatibility complex class I- and class II-binding peptides.</a> Immunology. 124 (1), 129-140 (2008).</li>
<li>Bergwelt-Baildon, M. S. von <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells%3A+potential+for+clinical+application%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99 (9), 3319-3325 (2002).</li>
<li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients%3A+analysis+of+immunogenicity+and+homing+potential%5BTitle%5D)+AND+%22Clin+Exp+Immunol%22%5BJournal%5D)">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155 (2), 249-256 (2009).</li>
<li>Schultze, J. L., Grabbe, S., von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((DCs+and+CD40-activated+B+cells%3A+current+and+future+avenues+to+cellular+cancer+immunotherapy%5BTitle%5D)+AND+%22Trends+Immunol%22%5BJournal%5D)">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25 (12), 659-664 (2004).</li>
<li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chromosomal+translocations+are+associated+with+poor+prognosis+in+chronic+lymphocytic+leukemia%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Chromosomal translocations are associated with poor prognosis in chronic lymphocytic leukemia.</a> Blood. 107 (2), 742-751 (2006).</li>
<li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma%5BTitle%5D)+AND+%22Gene+Ther%22%5BJournal%5D)">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15 (13), 955-965 (2008).</li>
</ol>

The experiments were performed in accordance with the national and European guidelines for laboratory animal keeping. Referring the German animal welfare law the animals were controlled regularly by the proper authorities.

Modifications of this protocol are possible, especially regarding the origin of CD40-stimulus and the type of mouse strain used, also yielding in efficient generation of murine CD40-activated B cells. Ahmadi et al. have shown similar results for mouse fibroblasts transfected with murine CD40-Ligand. In this context presentation of xeno-antigen can be excluded with certainty, but intensive studies regarding security and toxicity in vivo gave no signs for inflammation or auto-immunity reaction in this setting usin...

<table><tbody><tr><td colspan="4">A. Preparation of Media:</td></tr><tr><td colspan="4">1. Feeder cell wild type medium</td></tr><tr><td>RPMI 1640</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine, 300 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td>FBS, 10%</td><td></td><td></td><td></td></tr><tr><td>HEPES, 10 mM</td><td></td><td></td><td></td></tr><tr><td>Gentamycin, 15 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td colspan="4">2. Feeder cell selection medium</td></tr><tr><td>RPMI 1640</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine, 300 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td>FBS, 10%</td><td></td><td></td><td></td></tr><tr><td>HEPES, 10 mM</td><td></td><td></td><td></td></tr><tr><td>Gentamycin, 15 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td>Hygromycin B, 0.2 mg/mL</td><td></td><td></td><td></td></tr><tr><td colspan="4">3. mCD40-B washing medium</td></tr><tr><td>D-MEM</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine, 580 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td>Glucose, 4.5 mg/mL</td><td></td><td></td><td></td></tr><tr><td>HEPES, 10 mM</td><td></td><td></td><td></td></tr><tr><td>Gentamycin, 15 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td colspan="4">4. mCD40-B culture medium</td></tr><tr><td>D-MEM</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine, 580 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td>Glucose, 4.5 mg/mL</td><td></td><td></td><td></td></tr><tr><td>HEPES, 10 mM</td><td></td><td></td><td></td></tr><tr><td>Gentamycin, 15 &amp;mu;g/mL</td><td></td><td></td><td></td></tr><tr><td>MEM, 0.1mM (1x)</td><td></td><td></td><td></td></tr><tr><td>FBS, 10%</td><td></td><td></td><td></td></tr><tr><td colspan="4">B. Miscellaneous Reagents:</td></tr><tr><td>RPMI 1640</td><td>Invitrogen</td><td>REF 21875</td><td></td></tr><tr><td>D-MEM</td><td>Invitrogen</td><td>REF 41965</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s PBS (10x)</td><td>PAA Laboratories</td><td>Cat No H15-011</td><td></td></tr><tr><td>Gencin&amp;#174;</td><td>Delta Select</td><td>Art No 7395800</td><td></td></tr><tr><td>FBS</td><td>Lonza Inc.</td><td>Cat No DE14-802C</td><td></td></tr><tr><td>HEPES Buffer</td><td>PAA Laboratories</td><td>Cat No S11-001</td><td></td></tr><tr><td>MEM NEAA</td><td>Invitrogen</td><td>REF 11140</td><td></td></tr><tr><td>Hygromycin B</td><td>PAA Laboratories</td><td>Cat No P02-015</td><td></td></tr><tr><td>Recombinant Murine Interleukin-4</td><td>Immunotools</td><td>Cat No 12340045</td><td></td></tr><tr><td>Cyclosporin A</td><td>Novartis AG</td><td>Cat No NDC 0078-0109-01</td><td></td></tr><tr><td>Trypsin/EDTA (10x)</td><td>Invitrogen</td><td>REF 15400-054</td><td></td></tr><tr><td colspan="4">C. Miscellaneous Supplies:</td></tr><tr><td>Sterile pipette tips</td><td>Sarstedt Ltd</td><td></td><td></td></tr><tr><td>6-well plate</td><td>Nalge Nunc international</td><td>Cat No 140675</td><td></td></tr><tr><td>50mL conical tube</td><td>BD Biosciences</td><td>Cat No 352070</td><td></td></tr><tr><td>Tissue culture flask</td><td>Sarstedt Ltd</td><td>Cat No 83.1813.002</td><td></td></tr></tbody></table>

murine model of cd40-activation of 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.

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

Hi, my name is Tanya Wig and I'm a PhD student and working in the laboratory for tumor immunology and transplantation immunology at the University Hospital of Cologne. My studies mainly focus on marine CD 40 activated B cells and their ability to induce immune responses in vivo. Originally, human CD 40 B cells were generated as alternative antigen presenting cells to dendritic cells.Those cells are aimed to be used for immunotherapeutic purposes In clinical settings, CD 40 B cells are generated under CD 40 signaling and stimulation with IL four. According to this system, they can be expanded over several weeks. After optimizing the generation of human CD 40 B cells from PBMC in our lab, we transferred this principle successfully to mice and this is what I want to show you today.Starting from spleen, we generate marine CD 40 activated B cells within 14 days of cell culture. The main steps are the preparation of feeder cells, therefore we use marine CD 40 ligand transfected heer cell line, which is adherent and becomes irradiated with 78 gray and plated onto six well plates. Second, we start marine CD 40 DB culture by using freshly purified SPOC cytes from black six mice, which become treated with marine interleukin four beta macab to ethanol and cyclosporine A.Those cells are then transferred on irradiated feta cells on six well plates.This procedure is repeated every three to four days until day 14 of cell culture. To end up with highly pure marine CD 40 activated B cells, we begin the generation of marine CD 40 activated B cells using freshly purified SP cytes thromb LXi mice. First we wash the SP cytes by Resus suspending the cells in 50 milliliter of marine CD 40 B washing medium.Then spin down the cells for seven minutes at 1300 RPM corresponding 260 5G for determination of the cell count. Thoroughly resuspend the cytes in 10 milliliter of marine CD 40 B washing medium, and finally mix the cells by vortexing. Then determine the cell count.Spin down the required amount of cytes for seven minutes at 1300 RPM corresponding 260 5G using a six well plate. You need five times 10 to the six cells per well, thus 30 times 10 to the six cells per plate resource. Spend the required amount at one point 25 times 10 of the six cells per milliliter in marine CD 40 B culture media.Then freshly supplement the cell suspension with marine IL four in a concentration of one unit per milliliter for stimulation cyclosporine A in a concentration of oh 0.63 microgram per milliliter to inhibit outgrowth of T cells and 100 micromolar Beamer CAPTA ethanol. Now the cell suspension is ready for stimulation with feer cells. We are using marine CD 40 ligand transfected healer cells to stimulate cell survival differentiation and proliferation as the heer cell line can survive in culture for several months.Loss of marine CD 40 leg expression has to be excluded by fox regularly. CD 40 ligand transfected healer is an adherence cell line cultivated at 37 degrees with 5%CO2 in selection medium supplemented with icing B in a concentration of O 0.2 milligram per milliliter. For selection, this adherent epithelial cell line should never become completely confluent and should be split twice per week to split adherent cells.Aspirate the medium wash once with 10 milliliter of PBS. Gently swivel and add four milliliter trypsin EDTA in a 75 square centimeter flak. Incubate the flask for five to 10 minutes at 37 degrees in the incubator.Take the flask out again. Use gentle tapping to detach the cells from plastic. Then add 10 milliliter of biotype medium gently swivel and transfer the cells into a 50 milliliter tube.Spin down the feeder cells for seven minutes at 1300 RPM corresponding 260 5G. Carefully resuspend pelleted cells in 10 milliliter of wild type medium to determine the cell count for he a subculture RESO spent 2.5 times 10 to the six cells in 10 milliliter selection medium and to transfer them into a 75 square centimeter flask and incubate the feeder cells at 37 degrees in 5%CO2 for CD 40 stimulation. Take the rest of the heer cells and lethally, irradiate them with 78 grade to stop them from proliferation.Dilute the feeder cells in wild type medium with a cell density of O 0.2 times 10 to the six cells per liter and plate two milliliter per well on a six well plate. Let the cells become adherent again before using them as stimulator cells by incubating them for 24 hours in 37 degrees and 5%CO2 for stimulation of cytes with near CD 40 ligand. Check under the microscope if the feeder cells plated one day before are adherent.If the feeder cells are adherent carefully remove the SUP natin from each well then gently transfer four milliliter of the SPOC side suspension, which had been adjusted to one point 25 times 10 to the six cells per milliliter shortly before to each well for stimulation and incubate the plate at 37 degrees and 5%CO2 on day four subculture marine CD 40 B cells again for the first subculture of marine CD 40, activated B cells on day four and following subcultures every three to four days. Gently harvest the clustered cells from the six well plate by reus spending with a 10 milliliter pipe. Pool the marine CD 40 B cells in a 50 milliliter tube and spin down the cells for seven minutes at 1300 RPM corresponding 260 5G completely exchanged the medium by marine CD 40 B washing medium.And to determine the cell count of the marine CD 40 B cells, we suspend the marine CD 40 B cells at a cell density of oh point 75 times 10 to the six cells per milliliter in marine CD 40 B culture medium. Then again, supplement the cell suspension with fresh marine IL four in a concentration of one unit per milliliter, cyclosporine A in a concentration of oh point 63 microgram per milliliter and 100 micromolar beam up to ethanol. Of course, the irradiated feeder cells again have to be checked for the adherence under the microscope directly before use.If the feeder cells are adherent, carefully remove the SUP natin from each. Well then transfer the marine CD 40 B suspension prepared shortly before to each well for stimulation. The subculture is repeated identically after three to four days twice a week to end up with highly pure marine CD 40 activated B cells on day 14 of culture.At this point in time, Marine CD 40 B cells express co-stimulatory molecules such as CD 80, CD 86, and MHC molecules plus one and two, which can be checked by Fox during generation. The cells we generated right now can be used to do research on B-cell activation, differentiation and function, for example, to investigate their potential use for immunotherapeutic purposes.

murine-model-of-cd40-activation-of-b-cells

Research

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Lymphocytes are the major players in adaptive immune responses. Here, we present a lymphocyte purification protocol to determine the physiological functions of the desired molecules in lymphocyte activation in vitro and in vivo. The described experimental procedures are suitable for comparing functional capacities between control and genetically modified lymphocytes.

Isolation and Activation of Murine Lymphocytes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Here, we present a protocol for the in vivo determination of naïve CD4 T cell (T cell) activation, proliferation, and Th1 differentiation induced by GM-CSF bone marrow (BM)-derived dendritic cells (DCs). In addition, this protocol describes BM and T-cell isolation, DC generation, and DC and T-cell adoptive transfer.

An In Vivo Mouse Model to Measure Na&#239;ve CD4 T Cell Activation, Proliferation and Th1 Differentiation Induced by Bone Marrow-derived Dendritic Cells

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

We describe herein a simple analysis of the heterogeneity of the murine immune B cell compartment in the peritoneum, spleen, and bone marrow tissues by flow cytometry. The protocol can be adapted and extended to other mouse tissues.

Flow Cytometric Characterization of Murine B Cell Development

Learn CD40-activation of murine B cells from C57BL/6 splenocytes using IL-4 stimulation and feeder cell culture. This method enables generation of antigen-presenting cells for immunology research.

Murine Model of CD40-activation of B cells

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