Name: In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System (Video) | JoVE Journal
Uploaded: 2015-11-23T17:00:00.000+00:00
Duration: 8 min 18 s
Description: Learn an efficient method for generating plasmacytoid dendritic cells from common lymphoid progenitors using the AC-6 feeder system with Flt3 ligand. This technique enables large-scale pDC production for immunology research.

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5 minutes

We are grateful to Drs. Markus Manz and Irving Weissman for providing reagents. We also acknowledge the service provided by the Flow Cytometric Analysis and Cell Sorting Core Facility of the First and Second Core Laboratory at National Taiwan University College of Medicine and NTU hospital, respectively. This work was supported by the Ministry of Science and Technology, Taiwan (MOST 102-2320-B-002-030-MY3) and the National Health Research Institutes, Taiwan (NHRI-EX102-10256SI and NHRI-EX103-10256SI).

C57BL/6 wild-type mice were purchased from the National Laboratory Animal Center (NLAC), Taiwan. All mice were bred and kept under specific pathogen-free conditions. Protocols and animal use procedures were reviewed and approved by the Institutional Animal Care and Use Committee of National Taiwan University College of Medicine (Permit Number: 20120075). In addition, researchers made every effort to reduce the potential for pain, suffering, or distress in the animals while performing experiments. All procedures described were carried out at RT while wearing gloves.
1. Preparation of AC-6 Feeder cells
Note: The AC-6.21 (AC-6 in short) stromal cell line18 (provided by I. Weissman, Stanford University) should be maintained in RPMI supplemented with 15% heat-inactivated fetal bovine serum (FBS). To serve as feeder cells, AC-6 cells are &#947;-irradiated with 3,000 rad (30Gy) one day before the co-culture to prevent their proliferation. Note that AC-6 cells tend to lose their differentiation-supporting ability if left overcrowded during maintenance, or if their passage number is over 20.
<ol>
	<li>Wash AC-6 cells once with 1 ml Dulbecco&#39;s Phosphate-Buffered Saline (DPBS) and remove DPBS by aspiration. Treat AC-6 cells with 0.8 ml trypsin solution (0.05 % trypsin and 0.5 mM EDTA in DPBS) for 3-5 min at 37oC, 5% CO2 and then stop the reaction by diluting it with 10 volumes of culture medium to make single cell suspension.</li>
	<li>Seed AC-6 cells at 5.9x104 cells/well into 12-well plates and incubate at 37oC, 5% CO2 O/N. 
	Note: The density of AC-6 cells is very important as a lower density will favor cDC generation. Seeding at a density of 5.9x104 cells/well will allow AC-6 to reach confluency by the next day, which is optimal for derivation of pDCs from CLPs.</li>
	<li>Irradiate AC-6 cells at 3,000 rad (30 Gy) using &#947;-irradiator. 
	Note: The dose used to irradiate AC-6 is optimized to prevent the cells from proliferating and yet keep them viable long enough to provide the cytokines and cell-cell contact required for differentiation of DCs from CLPs.</li>
	<li>Aspirate medium and replace with 1 ml complete RPMI (RPMI with 10 % heat-inactivated FBS, 50 &#181;g/ml gentamycin and 50 &#181;M &#946;-mercaptoethanol).</li>
</ol>
2. Isolate BM Cells from Mice
<ol>
	<li>Sacrifice mice by CO2 asphyxiation and cervical dislocation. Place the mice on a dissection tray and sterilize with 70% ethanol. Make an incision at the mid-abdomen and remove the skin from the distal part of the mouse including the skin covering the lower extremities.</li>
	<li>Dissect mice in a semi-sterile hood. To release the femur and tibiae, clip the femur above and tibiae below the knee joint. Detach the muscles from the bones using scissors and place the bones in a 6 cm petri dish containing 5 ml complete RPMI.</li>
	<li>Move to a sterile culture hood. Fill a 3 ml syringe connected to a 27G needle with complete RPMI. Cut off both ends of the bones using scissors, and insert the 27G needle to flush the marrow out of the bones by gently injecting complete RPMI once from each end.</li>
	<li>Prepare a single cell suspension by gentle pipetting the cells up and down 3-5 times in the culture dish using the syringe without the needle.</li>
	<li>Centrifuge the cells for 5 min at 500 x g and decant the supernatant.</li>
	<li>Lyse red blood cells with 1 ml ACK buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) incubating for 1 min and stopping the reaction by diluting with 10 volumes of complete RPMI. Allow cells to stand for 5 min in order to pellet down dead cell clumps and tissue debris by gravity. 
	Note: Do not stand more than 5 min as this will result in cell loss due to settling of viable cells.</li>
	<li>Slowly decant cell-containing supernatant to a new tube leaving debris behind in the original tube.</li>
	<li>Centrifuge for 5 min at 500 x g to pellet the cells, then remove and discard the supernatant.</li>
	<li>Resuspend total BM cells (typically 4-6 x 107 BM cells are obtained from one mouse) in 100 &#181;l FACS buffer (1x PBS+ 2% FBS+ 1mM EDTA).</li>
</ol>
3. FACS Analysis and Sorting of CLPs
<ol>
	<li>Add anti-CD16/32 (hybridoma supernant clone 2.4G2, 50 &#181;l/reaction or 1-2 &#181;g/reaction) into cells in FACS buffer (step 2.9) for 1-2 min in order to block Fc receptors. 
	Note: Anti-CD16/32 is also called Fc block, which is added to prevent non-specific binding of antibodies to cells expressing Fc receptors including granulocytes, monocytes, and B lymphocytes.</li>
	<li>Simultaneously stain cells with the following fluorescent dye-labeled antibodies (for 4x107 cells) for 15 min on ice, avoiding ambient light. Antibodies: PE-conjugated lineage markers (0.2 &#181;g each) including anti-CD3 (17A2), anti-CD8 (53-6.7), anti-B220 (RA3-6B2), anti-CD19 (eBio1D3), anti-CD11b (M1/70), anti-Gr-1 (RB6-8C5), anti-Thy1.1 (HIS51), anti-NK1.1 (PK136), anti-TER119 (TER-119), and anti-MHC-II (NIMR-4), 0.2 &#181;g anti-c-Kit-PerCP-Cy5.5 (2B8), 1 &#181;g anti-Sca-1-FITC (D7), 0.4 &#181;g anti-M-CSFR-APC (AFS98), and 0.2 &#181;g anti-IL-7R&#945;-PE-Cy7 (A7R34).</li>
	<li>Wash cells with 3 ml FACS buffer, and centrifuge for 5 min at 500 x g.</li>
	<li>Resuspend the cells in 300 &#181;l FACS buffer and filter cells through a 40 &#181;M cell strainer.</li>
	<li>Wash the tube with an additional 100 &#181;l FACS buffer to recover any remaining cells and filter into a sterile FACS sorting tube using the same cell strainer.</li>
	<li>Perform flow cytometric analysis immediately after the staining using appropriate filters and voltages for signal detection. Use 488 nm laser to detect the FITC-, PerCP-Cy5.5-labeled antibodies, 561 nm laser to detect the PE- and PE-Cy7-labelded antibodies and 633 nm laser to detect the APC-labeled antibody.</li>
	<li>Sort out CLPs according to the following markers lin-c-kitintSca-1intM-CSFR-IL-7R&#945;+ (as stained in step 3.2) using a cell sorter. Collect the sorted cells in a 15 ml tube containing 8 ml of complete RPMI as a cushion.</li>
	<li>Record the absolute number of sorted cells as shown by the cell sorter at the end of the run. Typically, 5x104 CLPs can be obtained from the BM of one mouse.</li>
</ol>
4. Coculture CLPs and AC-6
<ol>
	<li>Centrifuge the sorted cells from step 3.7 for 10 min at 500 x g, remove the supernatant and resuspend the cell pellet with enough complete RPMI to obtain a cell density around 5x104 cells/ml. Seed 500 cells/well into the 12-well plate containing feeder cells prepared in step 1.4.</li>
	<li>Add 100 ng/ml FL19 (hu-Flt3L-Ig generated in-house using an expression system provided by M. Manz, University Hospital Z&#252;rich, Switzerland) and incubate at 37oC, 5% CO2 with periodic visual monitoring of DC development under a microscope. 
	Note: Commercially available recombinant human FL or mouse FL can be used as a substitute for hu-Flt3L-Ig to support DC development.</li>
	<li>Collect the cells in the supernatant at day 12 and wash the wells once with 0.5 ml complete RPMI medium combining the resulting supernatants. Add 0.5 ml fresh medium and scrap the adherent cells with a cell scraper.</li>
	<li>Combine the cell-containing medium from both parts and centrifuge for 5 min at 500 x g.</li>
	<li>Decant supernatant and resuspend cells in 50 &#181;l FACS buffer. Add 50 &#181;l anti-CD16/32 hybridoma supernatant and incubate for 1-2 min to block Fc receptors.</li>
	<li>Enumerate and stain all the cells with the following antibodies (0.05 &#181;g each) anti-CD11c-APC (N418), anti-CD11b-FITC, and anti-B220-PE.</li>
	<li>Wash and centrifuge the cells as described in step 3.3.</li>
	<li>Resuspend the cells in 100 &#181;l FACS buffer, gate on CD11c+ and analyze for cDCs (CD11c+CD11b+B220-) and pDCs (CD11c+CD11b-B220+)17.</li>
</ol>

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</ol>

Authors have nothing to disclose.

Here we describe an in vitro culture system for the robust generation of DCs, and pDCs in particular, from a small number of CLPs. The uniqueness of this culture system is due to the use of AC-6 cells, a stromal cell line, as feeders. This approach has been shown to provide not only the cytokines, such as IL-7, SCF, M-CSF and FL20, but also the cell-cell contact21 necessary to support DC development. AC-6 cells have been used previously to facilitate the study of in vitro DC develo...

Dendritic cells (DCs) are professional antigen-presenting cells that play an important role in controlling immune responses1. While DCs are heterogeneous, they can be broadly classified into two populations, conventional DCs (cDCs) and plasmacytoid DCs (pDCs)2,3. In addition to lymphoid organs, cDCs and pDCs are also found in tissues including lung, intestine, and skin2. The morphology of cDCs and pDCs differs, with cDCs exhibiting dendrite-like projections and the shape of pDCs being more plasma cell-like. In addition, the common mouse DC marker, CD11c, is more highly expressed on cDCs than on pDCs. Moreover, cDCs can be further divided into CD11b+CD24- cDCs and CD11b-CD24+ cDCs, both of which express higher levels of MHC class II and costimulatory molecules than do pDCs2. Mature pDCs, on the other hand, have been shown to selectively express Siglec-H and BST24. Functionally, cDCs are better antigen presenting cells than are pDCs; however, pDCs can produce a vast amount of type I interferon upon virus infection or inflammatory stimulation5.
Both cDCs and pDCs are short-lived, and therefore, must be constantly replenished from progenitors within the bone marrow (BM)6,7. Adoptive transfer of common myeloid progenitors (CMPs) and common lymphoid progenitors (CLPs) into lethally-irradiated mice demonstrates that cDCs and pDCs can be generated from both lineages8-10. However, there is a subset of pDCs that express RAG1/2 and possess rearranged D-J segments at the IgH locus11,12. These cells also share molecular similarities with B lymphocytes, including expression of the B220 marker, nucleic acid-sensing receptors (TLR7/TLR9), and transcription factors (Spib and Bcl11a)13, features all believed to be signatures of the lymphoid lineage. Therefore, CLPs may be good choices for in vitro generation of pDCs because of lineage similarity.
While the frequency of both cDCs and pDCs in humans and mice is very low6, DCs, particularly cDCs, can be generated in vitro from BM or progenitors in the presence of cytokines, such as GM-CSF11,14 or Flt3 ligand (FL) using feeder-free systems 11,15,16. Unfortunately, however, it is not possible to produce large numbers of pDCs in vitro using FL11,15,16. Previously we demonstrated that pDCs can be efficiently generated in vitro from CLPs using the AC-6 feeder system17. The advantage of using the AC-6 stromal cell line in the culture system is that it provides cell-cell contact and secretion of cytokines that support the generation of large numbers of pDCs from CLPs. Although production with this system is quite robust, careful replication of the procedures described below is required in order to ensure efficient generation of pDCs.

<table><tbody><tr><td>Anti-mouse Ly6g/Ly6c (PE), clone RB6-8C5</td><td>Biolegend</td><td>108408</td><td>linage marker</td></tr><tr><td>Anti-mouse NK1.1 (PE), clone PK136</td><td>Biolegend</td><td>108708</td><td>linage marker</td></tr><tr><td>Anti-mouse&amp;nbsp; CD11b (PE), cloneM1/70</td><td>Biolegend</td><td>101208</td><td>linage marker</td></tr><tr><td>Anti-mouse CD19 (PE), clone eBio1D3</td><td>Biolegend</td><td>115508</td><td>linage marker</td></tr><tr><td>Anti-mouse B220 (PE), clone RA3-6B2</td><td>Biolegend</td><td>103208</td><td>linage marker/FACS</td></tr><tr><td>Anti-mouse CD3 (PE), clone 17A2</td><td>Biolegend</td><td>100308</td><td>linage marker</td></tr><tr><td>Anti-mouse CD8a (PE), clone 53-6.7</td><td>Biolegend</td><td>100707</td><td>linage marker</td></tr><tr><td>Anti-mouse MHC-II (PE), clone NIMR-4</td><td>Biolegend</td><td>107608</td><td>linage marker</td></tr><tr><td>Anti-mouse Ter119 (PE), clone TER-119</td><td>Biolegend</td><td>116208</td><td>linage marker</td></tr><tr><td>Anti-mouse Thy1.1 (PE), clone HIS51</td><td>eBioscience</td><td>12-0900-83</td><td>linage marker</td></tr><tr><td>Anti-mouse M-CSFR (APC), clone AFS98</td><td>Biolegend</td><td>135510</td><td>FACS</td></tr><tr><td>Anti-mouse c-Kit (PerCP/Cy5.5), clone 2B8</td><td>Biolegend</td><td>105824</td><td>FACS</td></tr><tr><td>Anti-mouse Sca-1 (FITC), clone D7</td><td>Biolegend</td><td>108106</td><td>FACS</td></tr><tr><td>Anti-mouse IL-7Ra (PE/Cy7), clone A7R34</td><td>Biolegend</td><td>135014</td><td>FACS</td></tr><tr><td>Anti-mouse CD11c (PerCP/Cy5.5), clone N418</td><td>Biolegend</td><td>117328</td><td>FACS</td></tr><tr><td>Anti-mouse CD11b (FITC), clone M1/70</td><td>Biolegend</td><td>101206</td><td>FACS</td></tr><tr><td>FACSAria III</td><td>BD Biosciences</td><td></td><td>Cell sorter</td></tr><tr><td>FACS sorting tube&amp;nbsp;</td><td>BD Biosciences</td><td>352054</td><td></td></tr><tr><td>FlowJo</td><td>FlowJo LLC</td><td></td><td>Flow analysis sofrware</td></tr></tbody></table>

in vitro generation of murine plasmacytoid dendritic cells from common lymphoid progenitors using the ac-6 feeder system

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during inflammatory responses. Unfortunately, study of pDC function is hindered by their low frequency in lymphoid organs, and existing methods for in vitro DC generation predominantly favor the production of cDCs over pDCs. Here we present a unique approach to efficiently generate pDCs from common lymphoid progenitors (CLPs) in vitro. Specifically, the protocol described details how to purify CLPs from bone marrow and generate pDCs by coculturing with &#947;-irradiated AC-6 feeder cells in the presence of Flt3 ligand. A unique characteristic of this culture system is that the CLPs migrate underneath the AC-6 cells and become cobblestone area-forming cells, a critical step for expanding pDCs. Morphologically distinct DCs, namely pDCs and cDCs, were generated after approximately 2 weeks with a composition of 70-90% pDCs under optimal conditions. Typically, the number of pDCs generated by this method is roughly 100-fold of the number of CLPs seeded. Therefore, this is a novel system with which to robustly generate the large numbers of pDCs required to facilitate further studies into the development and function of these cells.

A total of 4-6x107 BM cells are typically isolated from femurs and tibiae of one 6-8 wk-old, wild-type C57BL/6 mouse. To sort out CLPs, total BM cells are stained with PE-conjugated antibodies against lineage markers (CD3, CD8, B220, CD19, CD11b, Gr-1, Thy1.1, NK1.1, TER119, and MHC-II), anti-c-Kit-PerCP/Cy5.5, anti-Sca-1-FITC, anti-M-CSFR-APC and anti-IL-7Rα-PE/Cy7, analyzed and sorted with a cell sorter. The sorting strategy for CLP is shown in Figure 1. Typically, 5x104 CLPs are ...

In this study we present an in vitro culture system that can efficiently generate pDCs by co-culturing common lymphoid progenitors with AC-6 feeder cells in the presence of Flt3 ligand.

Plasmacytoid dendritic cells (pDCs) are powerful type I interferon (IFN-I) producing cells that are activated in response to infection or during ...

The overall goal of this procedure is to provide an efficient method for generating plasma cy dendritic cells or PDCs for the study of their development. This method can help key questions in immuno fat field, such as How is PDC development regulated? The main advantage of this technique is that allows the use of a small number of progenitor cells to efficiently generate large quantities of PDCs.The implications of this technique stand toward the therapy of PDC associated autoimmune disease such as systemic lupus, athetosis, and psoriasis. Demonstrating the procedure today will be xang a great student from laboratory On the day before the bone marrow cell harvest seed AC six stromal cell line cells at 5.9 times 10 to the fourth cells per well into individual wells of 12 well plates and incubate the plates at 37 degrees Celsius and 5%carbon dioxide overnight. The next morning, gamma irradiate the AC six cells at 3000 rad and replace the culture medium with one milliliter of complete medium.Next place the appropriate number of adult wild type C 57 black six mice in a dissecting tray and sterilize the animals with 70%ethanol. Then move the tray to a semi sterile hood and to make incisions in the mid abdomens of each animal, removing the skin from the distal parts, including the lower extremities to release the femurs and tibiae, clip the femur above and the tibiae below the knee joints and use scissors to detach the muscles from the bones As they are collected. Place the bones in a six centimeter Petri dish containing five milliliters of complete RPMI and transfer the Petri dish to a sterile culture hood.Now fill a three milliliter syringe equipped with a 27 gauge needle with complete RPMI and use scissors to cut off both ends of each bone. Insert the needle into each bone and flush out the marrow with a gentle injection of complete RPMI once from each end. Then without the needle, gently aspirate and dispense the cells three to five times in the culture dish to generate a single cell suspension.After spinning down the cells lyce the red blood cells with one milliliter of a CK buffer, stopping the reaction with 10 volumes of complete RPMI. After one minute allow the dead cell clumps and debris to sediment for five minutes and then carefully decant the supernatant into a new tube. Then after spinning down the cells again, we suspend the total bone marrow cells in 100 microliters of fax buffer to analyze the common lymphoid progenitors or CLPs by flow cytometry.Next FC block the cells with anti CD 16 to 32 for one to two minutes, followed by simultaneous staining of the cells with the appropriate antibody cocktail. After 15 minutes on ice, wash the cells in three milliliters of fax buffer and resuspend the pellet in 300 microliters of fresh fax buffer. Then filter the cell suspension through a 40 micrometer strainer and immediately sort the cells on a flow cytometer using the appropriate filters and voltages.Collecting the cells of interest in a 15 milliliter tube, containing eight milliliters of complete RPMI. To generate a highly enriched PDCM population, it is a crucial to sort out the right population of CLPs from harvested bone marrow cell for their in vitro culture with a Z six feeder system. At the end of the sort, spin down the cells and resuspend the CLP cell pellet with enough complete RPMI to obtain a cell density of about five times 10 of the fourth cells per milliliter.Next seed 500 cells per well into the 12 well plate containing the AC six feeder cells and supplement the co cultures with 100 nanograms per milliliter FL three ligand, then incubate the cells at 37 degrees Celsius and 5%carbon dioxide with periodic visual monitoring of the dendritic cell or DC development under a microscope. On day 12, harvest the cells in the co-culture supernatant and wash each well one time with half a milliliter of complete RPMI medium, pooling the resulting snat and washes. Then add half a milliliter of fresh medium to each well and use a cell scrapper to detach the adherent cells.Combine the detached cells with the floating cells and centrifuge the resulting cell suspension. Resus suspending the pellet in 50 microliters of fax buffer. Then count the cells and stain them for CD 11 C, CD 11 b and B two 20 expression after FC blocking.As just demonstrated, the co-culture cells can then be analyzed for the presence of conventional CD 11 C, positive CD 11 B, positive B two 20 negative and plasmacytoid CD 11 C positive CD 11 B.Negative B two 20 positive DC populations a total of four to six times 10 to the seventh. Bone marrow cells are typically isolated from the femurs and tibia of one six to eight week old wild type C 57 black six mouse with five times 10 to the fourth CLPs commonly obtained from one C 57 black six mouse. After sorting by flow cytometry as just demonstrated by days three to four of co-culture with AC six feeder cells, cobblestone area forming cells begin to appear with round shaped Plasmacytoid DC appearing suspended on top of the cobblestone area forming cells.By day eight, the number of plasma cyto DC peaks around days 12 to 14, at which point the cells start to undergo apoptosis following their full development and their numbers begin to gradually decrease. Conventional DC colonies appear later at day six to eight and are adherent with a larger spindle like morphology. Flow cytometric analysis demonstrates that the co cultures are 91%CD 11 C, positive CD 11 B, negative B two 20 positive plasmacytoid dc, and 6%CD 11 C, positive CD 11 B, positive B two 20 negative conventional DC revealing a 100 fold expansion of plasmacytoid DC numbers from the original CLP population by this method.While attempting this procedure, it is important to make sure that the a c six cell is in good condition and the C density is correct before adding the clp after a development. This technique paved the way for researchers in DC biology to overcome the limitation of using small number of projectors to study DC development from different projectors. After watching this video, you should have a good understanding of how to generate PCs in vitro from CLP using a C six BEAT system.

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Learn an efficient method for generating plasmacytoid dendritic cells from common lymphoid progenitors using the AC-6 feeder system with Flt3 ligand. This technique enables large-scale pDC production for immunology research.

In Vitro Generation of Murine Plasmacytoid Dendritic Cells from Common Lymphoid Progenitors using the AC-6 Feeder System

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