Name: Generation of Human CD40-activated B cells (Video) | JoVE Journal
Uploaded: 2009-10-16T23:30:00.000+00:00
Duration: 13 min 27 s
Description: Learn to generate human CD40-activated B cells from peripheral blood mononuclear cells using CD40 ligand and interleukin-4 stimulation. This method enables expansion of potent antigen-presenting cells for immunology research.

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5 minutes

The protocol for the generation of human CD40-activated B cells from PBMC is divided into two parts: Part A demonstrates the preparation of CD40 ligand expressing NIH/3T3 cells, which will be used as plate-bound feeder cells. Part B describes the actual CD40-B culture.
A. Preparation of feeder cells (tCD40L NIH/3T3)
The tCD40L NIH/3T3 is an adherent murine fibroblast 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 Trypsin/EDTA in a 75 cm2 flask for 5-10 minutes at 37°C. Use gentle tapping to detach the cells.</li>
 <li>Add 10 mL of wild type medium and swivel gently.</li>
 <li>Transfer the cell suspension into a 50 mL tube with a sterile pipette and spin the cells down at 225 x g for 5 min.</li>
 <li>Remove the supernatant and resuspend the 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>1.5 x 106 cells for subculturing</li>
 <li>0.2 x 106 cells/well for irradiation used for the CD40-B cell culture</li>
 <li>remainder to freeze (if needed).</li>
 </ol>
 </li>
 <li>Spin the cells down at 225 x g for 5 min.</li>
 <li>Remove the supernatant.
 <ol>
 <li>For subculturing: resuspend 1.5 x 106 cells in 10 mL wild type medium in a 75 cm2 cell culture flask (cell density 1.5 x 105 cells/mL), add G-418 [0.7 mg/mL] and incubate the cells at 37°C with 5 % CO2. Split the cells twice per week.</li>
 <li>For the CD40-B cell culture: You need 1.2 x 106 cells for one 6-well plate. Resuspend the cells in wild type medium at a density of 0.1 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 tCD40L NIH/3T3 cells are adherent (at least 4 hours: check adherence with the microscope; do not wait more than 24 h to start B-cell stimulation). (Continue with B.)</li>
 </ol>
 </li>
</ol>
B. CD40-B cell culture
I. Preparation of PBMCs for CD40-stimulation (day 0):
Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of interleukin-4 and cyclosporin A to the growth medium immediately before use.
<ol>
 <li>Take PBMCs, either fresh or appropriately thawed. Resuspend PBMCs twice in 50mL of 1x PBS to wash them and spin down first time at 265 x g for 7 min and a second time at 190 x g for 7 min to remove other cells. Discard supernatant and resuspend the cells in 20 mL of PBS. Determine the cell number in an aliquot of the cell suspension.</li>
 <li>Spin down required amount of cells at 225 x g for 5 min. For a 6-well plate 4 x 106 cells / well are needed, thus 24 x 106 cells per plate.</li>
 <li>Remove the supernatant and resuspend the PBMC at 1 x 106 cells/mL in CD40-B culture medium freshly supplemented with 50 U/mL of interleukin-4 as a growth factor and 0.63 μg/mL cyclosporine A to prevent outgrowth of T-cells (Given concentrations refer to one mL culture medium!).</li>
 <li>Remove the supernatant from 6-well plate pre-incubated with tCD40L NIH/3T3 cells.</li>
 <li>Wash the adherent tCD40L NIH/3T3 cells with 2 mL PBS per well first and in a second step with 2 mL of CD40-B washing medium.</li>
 <li>Gently add 4 mL of PBMC suspension (1 x 106 cells/mL) to each well of the 6-well plate.</li>
 <li>Incubate the cells at 37°C with 5 % CO2.</li>
 <li>On day 7, reculture the cells (Continue with 3.2.).</li>
</ol>
II. Recultivation of CD40-B cells (day 7 and then every 3-4 days):
<ol>
 <li>Harvest clusters of CD40-B cells from 6-well plate by resuspending with a 10 mL pipette and pool them into a 50 mL tube.</li>
 <li>Spin down at 225 x g for 7 minutes and completely replace the supernatant with CD40-B washing medium. While counting the cell amount of an aliquot, spin the cells down at 225 x g for 5 minutes. Resuspend CD40-B cells in CD40-B culture medium at a concentration of 1 x 106 cells/mL.</li>
 <li>Add fresh solutions of interleukin-4 in a concentration of 50 U/mL and 0.63 μg/mL cyclosporine A to the medium.</li>
 <li>Remove the supernatant from a 6-well plate pre-incubated with tCD40L NIH/3T3 cells.</li>
 <li>Wash the adherent cells with 2 mL PBS per well first and in a second step with 2 mL of CD40-B washing medium.</li>
 <li>Gently add 4 mL (1 x 106 cells/mL) of CD40-B suspension to each well of the 6-well plate.</li>
 <li>Incubate plates at 37°C with 5 % CO2.</li>
 <li>Subculture cells again every 3-4 days to end up with highly pure human 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>The plate-bound feeder cells used for the stimulation should not be older than 24h?</li>
 <li>Is a contamination with mycoplasma possible?</li>
 <li>Was the interleukin-4 solution used for supplementation freshly thawed and had the appropriate biological activity?</li>
 <li>Was cyclosporin A added in the correct concentration?</li>
</ol>
<img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="Flow cytometry analysis: CD markers, fluorescence intensity; graphs show immune cell activation study." data-altupdatedat="1787630408000" data-alt="Figure 1"> Figure 1. Please <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">click here</a> for a larger version of figure 1.
<img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="Flow cytometry FACS diagram: SSC vs FSC plot, CD154 expression histogram, fluorescence intensity." data-altupdatedat="1787630408000" data-alt="Figure 2"> Figure 2. Please <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">click here</a> for a larger version of 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, 2757-2765 (1997).</li>
<li>Schultze, J. L., Grabbe, S., vonBergwelt-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, 659-664 (2004).</li>
<li>Bergwelt-Baildon, M. von <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, 2786-2789 (2006).</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, 1464-14 (2008).</li>
<li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</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, 2836-2843 (2003).</li>
<li>von Bergwelt-Baildon, M. S. <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, 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, 249-256 (2009).</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, 955-965 (2008).</li>
</ol>

<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>DMEM-Ham&amp;rsquo;s / F12</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine (365 &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>DMEM-Ham&amp;rsquo;s / F12</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine (365 &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>G-418 (0.7 mg/mL)</td><td></td><td></td><td></td></tr><tr><td colspan="4">3. CD40-B washing medium</td></tr><tr><td>IMDM</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine (584 &amp;mu;g/mL)</td><td></td><td></td><td></td></tr><tr><td>HEPES (25 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. CD40-B culture medium</td></tr><tr><td>IMDM</td><td></td><td></td><td></td></tr><tr><td>L-Glutamine (584 &amp;mu;g/mL)</td><td></td><td></td><td></td></tr><tr><td>HEPES (25 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>rh Transferrin (50 &amp;mu;g/mL)</td><td></td><td></td><td></td></tr><tr><td>rh Insulin (5 &amp;mu;g/mL)</td><td></td><td></td><td></td></tr><tr><td>AB-Humanserum (10%)</td><td></td><td></td><td></td></tr><tr><td colspan="4">B. Miscellaneous Reagents:</td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td>DMEM / Ham&amp;rsquo;s F12</td><td>PAA Laboratories</td><td>Cat No: E15-813</td><td></td></tr><tr><td>IMDM</td><td>Invitrogen</td><td>REF 21980-032</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>AB-Human Serum</td><td>Invitrogen</td><td>Cat No 34005100</td><td></td></tr><tr><td>holo-Transferrin human</td><td>Sigma-Aldrich</td><td>Cat No T0665</td><td></td></tr><tr><td>Insulin human</td><td>Sigma-Aldrich</td><td>Cat No I2643</td><td></td></tr><tr><td>Gencin&amp;#174;</td><td>Delta Select</td><td>Art No 7395800</td><td></td></tr><tr><td>Recombinant Human Interleukin-4</td><td>Immunotools</td><td>Cat No 11130045</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>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>G-418 Sulphate</td><td>PAA Laboratories</td><td>Cat No P02-012</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></td><td></td><td></td><td></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>

generation of human cd40-activated b cells

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.

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

Hi, my name is Alexander Shiku Van Hagen. I'm a member of the Laboratory for two Immunology and Transportation Immunology at the University Hospital of Cologne. In this video, we are going to show you how we can generate human CD 40 activated B cells from peripheral blood mononuclear cells.One of the main interests of our laboratory is the study of the antibody independent functions of BB cells. Beyond their role as producers of antibodies, B cells have an important role as antigen presenting cells both under physiologic conditions as well as in autoimmune disease. After activation, B cells can acquire potent antigen presenting and immunostimulatory capacities, especially when activated via CD 40.CD 40. Activated B cells have several advantages when compared to dendritic cells. They can be easily expanded at relatively low cost from small amounts of peripheral blood, even from cancer patients.Now our PhD student, Tanya Leic and our technician LER, will give you a step by step introduction on how to generate human CD 40 activated B cells from peripheral blood. Hello, my name is Tanya Livi. I'm a PhD student and I want to demonstrate to you how to generate human CD 40 activated B cells.This procedure was optimized in our lab. The main steps are, first the preparation of feeder cells. Therefore, we use human CD 40 ligand transfected N IH 33 cells, which become irradiated with 78 gray and plated onto six well plates.Second, we start the culture of human CD 40, activated B cells from purified PBMC, which become treated with human interleukin four and cyclosporine aim, those cells are then plated onto feta cells on six well plates. This procedure is repeated on day seven and then every three to four days until day 14 of cell culture. To end up with highly pure human CD 40 activated B cells, we start the generation of CD 40 activated B cells with freshly purified or appropriately thought human PBMC.First, we wash the pbmc for two times, therefore re suspend the cells in 50 milliliter of PBS. Spin down the PBMC for seven minutes at 1300 RPM corresponding 260 5G. This car, the supinate and grease, suspend the cells again in 50 milliliter of PBS spinned down again for seven minutes at 1100 RPM corresponding 190 G.This card the supinate again for determination of the cell count reus.Suspend the PBMC in 20 milliliter of PBS thoroughly mix the cells by vortexing and determine the cell count. Spin down the required amount of PBMC for CD 40 B culture for five minutes at 1200 RPM corresponding 220 5G. Using a six well plate, you need four times 10 to the six cells per well plus 24 times 10 to the six cells per plate.Then re suspend the PBMC at one times 10 to the six cells per milliliter in CD 40 B culture medium. Supplement the cell suspension with fresh human IL four in a concentration of 50 units per milliliter for stimulation and cyclosporine A in a concentration of oh point 63 microgram per milliliter to inhibit outgrowth of T-cell. Now the PBMC suspension is ready for stimulation with CD 40 ligand.To our knowledge, there is currently no functional GMP grade SOMAL CD 40 ligand available. Therefore, we use human CD 40 ligand transfected NIH three T three cells to stimulate cell survival differentiation and proliferation as the NIH three T three cell line can survive in culture for several months. Loss of CD 40 ligand expression has to be excluded by facts regularly.CD 40 ligand transfected, NIH three three is an adherence cell line and cultivated at 37 degrees with 5%CO2 in selection medium, which is supplemented with G 4 1 8 in a concentration of oh 0.7 milligram per milliliter. This adherent fibroblast cell line should never become completely confluent and should be split twice per week To split the adherence cells aspirate the medium wash once with 10 milliliter PBS gently swivel. Aspirate the medium again and add four milliliter tripsin EDTA in a 75 square centimeter flask.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 the wild type medium gently swivel and transfer the cells into a 50 milliliter tube. Spin down the NA eight three T three cells for five minutes at 1200 RPM corresponding to hundred and 20 5G. Carefully resuspend the paled cells in 10 millimeter wire type medium.Determine the cell count for N IH three three subculture and resuspend 1.5 times 10 to the six feeder cells in 10 milliliter wildtype medium. Transfer them in a 75 square centimeter flask and add G 4 1 8 in a concentration of oh 0.7 milligram per milliliter to select for the transfected cells which carry neomycin resistance. Genes finally incubate the feeder cells at 37 degrees in 5%CO2 for CD 40 B stimulation.Take the rest of the NI 8 3 3 cells and lethally, irradiate them with 78 grade to stop them from proliferation. Dilute the irradiated feeder cells in wire type medium with a cell density of oh 0.1 times 10 to six cells per milliliter plate two milliliter per well on a six well plate. Let the cells on six well plates become adherent again before using them as feeder cells by incubating them for 24 hours in 37 degrees and 5%CO2 for stimulation of bi lymphocytes with CD 40 ligand.Check under the microscope if the feeder cells plated one day before are adherent. If the feeder cells are adherent, remove SUP natin from each well and quickly wash them two times. First, wash each well with two milliliter PBS and second with two milliliters.CD 40 B washing Medium For stimulation at the PBMC suspension. Prepared shortly before with a cell density of one times 10 to the six cells per milliliter to the feeder cells. To do so, gently transfer four milliliter of the cell suspension to each.Well incubate the plate at 37 degrees in 5%CO2 on day seven. CD 40 B cells can be sub cultivated for the first subculture of CD 40. Activated B cells on day seven and following subcultures every three to four days.Gently harvest the clustered cells from the six 12 plate by Resus, suspending them with a 10 milliliter pipe. Pull the CD 40 B cells in a 50 milliliter tube. Spin down the CD 40 B cells for seven minutes at 1200 RPM corresponding 220 5G completely.Exchange the medium by CD 40 B washing medium and determine the cell count of the CD 40. Activated B cells. Re suspend the CD 40 B cells at one times 10 to the six cells per milliliter in CD 40 B culture medium.Then again, supplement the cell suspension with fresh human IL four in a concentration of 50 units per milliliter and cyclosporine A in a concentration of oh 0.63 microgram per milliliter for stimulation. Uc, irradiated feeder cells prepared one day earlier. Of course, the irradiated feeder cells again have to be checked for adherence under the microscope directly before use.If adherent, wash the feeder cells quickly for two times like before one time with PBS. The other time with CD 40 B washing media then carefully played four milliliter of the CD 40 B suspension adjusted to one times 10 to the six cells per milliliter on each well of the six well plate. The subculture is repeated identically after three to four days twice a week to end up with cell clusters of highly pure human CD 40 activated B cells on day 14 of culture.At this point in time, CD 40 B cells express co-stimulatory molecules such as CD 80, CD 86, CD 83 MHC molecules, and the so-called lymph node homing triad of CD 62 ligand CCR seven and LFA one, which can be checked by Fox during generation For troubleshooting. Please refer to to text version of this protocol. We hope that in the future CD 40 activated B cells will serve as an important tool for cancer immunotherapy.Good luck with your experiments.

generation-of-human-cd40-activated-b-cells

Research

JoVE Journal

Biology

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Generation of Human CD40-activated B cells

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