In this article, we provide a detailed protocol for the expression of fluorescent fusion proteins in murine bone marrow derived dendritic cells and macrophages. The method is based on the transduction of bone marrow progenitors with retroviral constructs followed by differentiation into macrophages and dendritic cells in vitro.
This method can help answer key questions in the fields of macrophage and dendritic cell biology about the sub-cellular localization and dynamics of various proteins in these cell types. The main advantages of this technique are that it is relatively simple, inexpensive, and demonstrates a better efficiency and stability of expression than the majority of other available methods. Demonstrating the procedure will be Jarmila Kralova, a grad student from my laboratory.
To produce the retrovirus, plate a single-cell suspension of Platinum Eco-packaging cells in a 10 centimeter petri dish and cultivate the cells in 15 milliliters of DMEM, supplemented with 10%fetal bovine serum, or FBS, until the culture is 50 to 60%confluent. The next day, add 20 micrograms of the retroviral construct of interest and 10 micrograms of PCL eco-packaging vector to 1 milliliter of DMEM without serum, with gentle mixing. Add 75 microliters of PEI in 1 milliliter of DMEM without serum and antibiotics to a second tube for a five minute incubation at room temperature, and mix the contents of both tubes together for an additional 10 minute incubation at room temperature.
Next, carefully replace the medium on the Platinum Eco cells with 8 milliliters of fresh 37 degree Celsius DMEM supplemented with 2%FBS and carefully add the transfection mixture in drops to the plate. After a 4 hour incubation at 37 degrees Celsius, replace the supernatent with 10 milliliters of 37 degree Celsius DMEM, supplemented with 10%FBS for a 24 hour incubation at 37 degrees Celsius. The next day, use a 5 milliliter pipette to transfer the ecotropic retroviral particle-containing supernatent to a 15 milliliter centrifuge tube and remove the debris and containing cells by centrifugation.
Meanwhile, add 10 milliliters of 37 degrees Celsius DMEM plus 10%FBS to the culture dish for another 24 hour incubation at 37 degrees Celsius and collect a second round of viral particles as just demonstrated. To harvest the bone marrow, in a tissue culture hood, use tweezers and scissors to remove the skin and part of the muscles from the hind limbs and carefully dislodge the acetabulum from the hip joint without breaking the femur. Cut the paws at the ankle joints and spray the bones with 70%ethanol.
Then remove the rest of the muscle with a paper towel and place the bones in a 5 centimeter petri dish of PBS supplemented with 2%FBS. Next, bend the knee joint of each bone set and carefully separate the bones with scissors. Use the scissors to remove about one to two millimeters of each epiphysis of one bone and use a two or five milliliter syringe equipped with a 30 gauge needle loaded with fresh PBS plus FBS to flush the bone marrow from both ends of the bone into a 15 milliliters centrifuge tube.
When the marrow has been collected from each of the bones, pellet the bone cells by centrifugation and re-suspend the pellet in 2.5 milliliters of red blood cell lysis buffer for two to three minutes at room temperature. After lysing, filter the cells through a 100 micrometer cell strainer into a new 15 milliliter centrifuge tube and restore the tenacity with 12 milliliters of PBS plus FBS. Re-suspend the pellet in DMEM, supplemented with 10%FBS and antibiotics for counting and plate five to 10 times 10 to the sixth bone marrow cells in a 10 centimeter, non-tissue culture treated petri dish in 10 milliliters of DMEM supplemented with serum and macrophage colony stimulating factor, or MCSF.
On day five of culture, carefully tilt the cell culture dish and remove all but approximately 5 milliliters of medium from the surface near the edge of the dish. Then add 20 milliliters of fresh 37 degree Celsius medium supplemented with FBS and MCSF to the dish and return the cells to the cell culture incubator. To harvest the cells, on day six to seven, remove all of the medium and floating cells and wash the culture with 37 degree Celsius PBS.
Detach the adherent cells with five milliliters of 0.02%EDTA and PBS for three to five minutes at 37 degrees Celsius in the tissue culture incubator and use a five milliliter pipette to gently flush the dissociated cells from the plate bottom. Then transfer the cells to a 50 milliliter centrifuge tube containing 25 milliliters of fresh PBS. To induce EGFP tagged protein expression in bone marrow derived antigen presenting cells, at the beginning of the cell differentiation protocol, dilute the bone marrow cells to a two to five times 10 to the sixth cells per milliliters of DMEM plus FBS and antibiotics conentration and plate the cells of one milliliter of medium per well, supplemented with the appropriate differentiation cytokine.
After four to six hours in the cell culture incubator, add two milliliters of the first round of collected virus supplemented with polybrene and transduce the cell cultures by centrifugation and a four hour incubation in the cell culture incubator. At the end of the transduction period, transfer the nonadherent cells from each well into individual 15 milliliter centrifuge tubes and collect the cells by centrifugation. If larger amount of cells is needed, the cells can be infected with the same constract in multiple wells and pooled before differentiation.
Then re-suspend the pellets in 10 milliliters of culture medium supplemented with the appropriate differentiation cytokine and plate the cells onto one 10 centimeter non tissue culture treated petri dish per tube, for their six to eight day culture, as just demonstrated. Evaluation of the efficacy of Platinum Eco transfection by flow cytometry after the collection of the second virus containing supernatent reveals a mean transfection efficiency of 62%for PSTPIP2-EGFP and 53%for OPAL1-EGFP. Retroviral transduction does not affect the differentiation status of the bone marrow derived cells with more than 90%of the cells in both cultures demonstrating positivity for their respective markers after transduction with either virus.
And with the appropriate, characteristic morphological changes observed for both types of differentiated cell. Of note, the overall expression of EGFP was lower in bone marrow derived dendritic cell cultures compared to bone marrow derived macrophage EGFP expression, regardless of type of retrovirus. While attempting this procedure, it is important to remember to use non tissue, culture treated plastic dishes that are typically used for cultivating bacteria if you plan to remove cells from the dish for experiment.
Following this procedure, other methods live cell imaging, super resolution microscopy or other types of microscopy can be employed to answer additional questions about the sub-cellular localization and dynamics of dendritic cell and macrophage proteins of interest. This technique paved the way for researchers in the immunology field in exploring the distribution and spacial relationships of proteins expressed by macrophages and dendritic cells and in furthering our understanding of the function of these protein during immune responses. Don't forget that working with viral particles can be extremely hazardous and that precautions, such as minimalizing aerosol generation, wearing protective equipment, and using a laminar flow hood, should always be taken while performing this procedure.
