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

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.