Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Preparation of dendritic cells from bone marrow provides an excellent source of antigen presenting cells that can be used for in vitro or in vivo experiments in two botton imaging of the immune system. The introduction of antigen presenting cells allows for the study of antigen-specific cell cell interactions in the context of the living lymph node. Here we demonstrate the sterile isolation of bone marrow and culture techniques for the production of mature dendritic cells.

Furthermore, we follow the development of dendritic cells from day one to day 11. This development's depicted with still images and in incubator videos made using scent technologies incus site. Finally, we show bone marrow derived dendritic cells presenting cove antigen to OT two T cells in the mirroring lymph node.

Hi, I'm Melanie Matthew in the laboratory of Micah Halen at the University of California nearby. Have you ever wondered what's going on inside during prober while your cells are growing? Today?

As part of our protocol, we're going to show you how to use S some instruments in site. The inky site allows you to track cell culture growth in your incubator. In this protocol, we're going to show you how to isolate and prepare bone marrow derived and group cells from mouse.

So the first thing I'm going to show you how to do is remove femur bones and isolate the bone marrow First pin down the animal. Next, wet the animal with 70%ethanol. Make your first incision along the midline of the animal.

Then cut inside the leg as I'm showing you here. To expose the muscle tissue above the femur, you should be able to pull aside the muscle and grasp the large femur bone with your dissection tweezers. As you're holding the bone with your dissection tweezers, move down the bone and make your first cut just below the joint.

Then work your scissors up the length of the femur and make it cut as close to the body as possible. This may result in a little bit of blood, especially if you cut the femoral artery. When you're removing the femur, it is extremely important that the bone is kept intact.

As you can see here at the ends of the bone have not been cut or disrupted. Once the femur has been removed, clip off any additional tissue and place the femur in a small dish of our PMI media. When removing additional tissue, be sure that the femur bone does not dry out.

At this point, the femur bones should be transferred into a dish containing 70%ethanol and placed on ice for five to 10 minutes. For the purpose of this video, we will continue on the lab bench. However, at this stage of the experiment, everything should be transferred into a tissue culture hood.

Once the bones have been soaked in ethanol, you can move them into a larger dish dish of sterile media for the removal of bone marrow. Now we are ready to remove and resuspend the bone marrow. Fill an insulin syringe with sterile media and placed to the side for later use.

Grasp the bone with your sterile tweezers. Hold it over a small plate for discarded pieces and cut the epiphysis or the ends of the bone off each side. Place your insulin syringe into one side of the bone and flush the marrow out of the bone into 15 mils of media.

Wash the bone out one or two more times until all of the marrow is flushed out. Once the marrow is flushed out of the bone, it will be almost completely white. Place the clean bone into the discard dish and repeat the process of the other femur.

Once this has been completed, gently disrupt any marrow pieces that were not broken apart by the washing process to create a single cell suspension. This will take five to 10 minutes of pipetting. Place a single cell suspension in a 50 mil conical tube, wash the plate at least three times with 10 mils of media.

Each time, spin down the cells, remove the media and resuspend the cells by firmly and briskly brushing your fingers against the bottom of the tube. To remove the red blood cells or RBCs, we perform a water lysis. Another method for red blood cell removal is an ammonium chloride lysis.

Both methods have been described in previous JoVE video articles. Isolation of CD four positive T-cells from mouse lymph nodes using the Milan max purification JoVE issue nine and the preparation of T-cell growth factor from rat cytes JoVE. Issue 10.

After removal of your RBCs, you are now ready to culture yourselves. So before I take you through dendri cell culture, I'd like to introduce you to the incus site. The incus site will allow subtracting predict cell culture growth within the incubator so we can watch ourselves on how they behave or misbehave.

Once a culture is set up, you place it in the appropriate size tray and slide the incus site shut. Then on your computer, you simply set the time interval and then choose the wells you would like image and let the inky site collect data for you. The movies will show you in this protocol, demonstrate dendritic cell morphology changes in vitro.

These videos were exported directly from the Incus site program. As the dendritic cells is developed from day one to day 11, you can see a true morphology change as they enlarge and take on the dendritic cell phenotype. After lysing, RBCs and removing them, cells are washed in primary dendritic cell media and plated at 5 million per mil.

These cells should be initially cultured in primary dendritic cell culture media, and later we culture them in secondary DC media, which is supplemented with 40 nanograms per mil IL four and a hundred nanograms per mil TNF alpha. These cytokines will aid in the development of mature dendritic cells that resemble monocyte derived dendritic cells. So now I'm going to show you what the cultures look like as they develop and how to maintain them over time.

Once you have plated the cells, you can culture the dendritic cells for 72 hours before your first media change. Here's what your typical culture will look like just after you plate the cells at day one. And here's a look at the cells.

On day three, you can see that cells have begun to adhere to the plate but have not taken on the classic dendritic cell phenotype. On day four, remove media and add 10 mils of fresh primary DC media aspiration of the media. On day four, we'll remove lymphocytes in any other non-adherent cells.

Now you can place yourselves back in the incubator for another three days, seven days after your initial plating. Here's what your typical culture will look like. You can tell that it's time to split the cells and repl them.

On day seven, the dendritic cells are removed from the plate by aspirating the media and then adding 300 millimolar sterile EDTA and incubating this for five minutes. Then the plate is repeatedly rinsed by holding at a 45 degree angle and pipetting the EDTA solution against the back of the plate. This is done for several minutes to ensure that you remove all adherent dendritic cells.

Cells are collected in a 50 milli conical tube and washed twice with secondary DC media. The cells are then resuspended and plated at 5 million cells per plate. Freshly replated day seven dendritic cells rapidly settle to the bottom of the plate, adhere and begin to spread out.

As you can see here in the Zichy site video, this video was taken in the incubator over the course of two days to show you the dendritic cell development from the initial replating done on day seven to early day nine. Morphology dendritic cells can be activated in antigen pulse by adding two micrograms per LLPS here with a hundred micrograms for ova for use with T-cell purified from OT two mice, which have ova specific CD four positive T-cells. If you're using a different antigen, it's important to make sure that you use an optimal concentration of antigen to pulse your dcs here, the OV an LPS are added to primary media such that the final concentration of LPS will be two micrograms per mil in 20 mils, and ova will be a hundred micrograms per mil in 20 mils.

Here's a photograph of what a typical day 10 culture of dendritic cells looks like. You can see in this photograph that the morphology is very typical of an antigen presenting cell. You should always check the percentage of CD 11 C positive dendritic cells by fax.

This is to ensure that you have a relatively pure culture of dendritic cells for use in your experiments. Here we use the inky site from SEN instruments to monitor the activation of dendritic cells that were LPS treated in antigen pulsed on day 10. This video spans a 24 hour period immediately after the addition of LPS and the val and peptide, and in it dendritic cells become more active and there are some obvious morphological changes.

Once your dendritic cells have been activated in antigen pulse, you can again collect them by incubation with 300 millimolar EDTA and washing the plate. Now spin your cells down, wash twice with 50 mils of RPMI media to remove any additional antigen and label them with the appropriate cell tracker dye. In this experiment, I will be imaging the cells in the inguinal lymph node.

We will need to inject two to 5 million dendritic cells into dermally into the flank region of the mouse shown here. We then allow 24 hours for the dendritic cells to home to the training lymph node. Here in this video, dendritic cells are labeled with CMF two hc, a blue cell tracker dye.

You can see the dendritic cells interacting with labeled T cells that are antigen specific for volin. These T cells are labeled with ccf SE.You can also use C-M-T-M-R or any one of the other cell tracker dyes for molecular probes. This is the early activation stage of T-cells interacting with dcs.

Because they're antigen specific, you can see that the T-cells are sticking to the tendrils of the blue labeled dcs. This concludes our protocol demonstrating the preparation and use of mouse derived bone marrow dendritic cells in a two botton imaging experiment. Thanks for watching and good luck with your own experiments.