This protocol helps in culturing bone marrow derived macrophages from iNOS or BH4 deficient mice using carefully defined media and serum with low levels of BH4 and nitrate to avoid interference with redox mechanisms. By carefully controlling the levels of potential contaminants in the culture process, we can limit nitric oxide interference, ensuring accuracy and reproducibility of the model system. This method allows for accurate measurements of biopterins, nitric oxide, and any downstream factors related to these essential compounds.
Demonstrating the procedure will be Dr.Thomas Nichol, a post-doctoral researcher from our lab. To begin, place the legs in 70%ethanol for two minutes to wash off any fur or residue. Then, transfer the legs to a clean, sterile bacteriological Petri dish.
Carefully scrape along the length of the bones with the blade, cutting through tendons and removing the muscle from the bone. Once the bones have been cleared of muscle, cut through the epiphysis at the top of the tibia, just below the knee. The tibia can be cut through at the bottom end, just above the intersection with the fibula.
Finally, cut through the epiphysis at either end of the femur, just short of the knee and hip joints. To flush the bone marrow, fill a 10 milliliter syringe with 10 milliliters of PBS and attach it to a needle. Then insert the needle into the medullary cavity of the bone.
Gently flush with one to two milliliters of PBS, running the needle up and down. Then, draw the suspension five to ten times in and out of a one milliliter syringe to disaggregate. Collect the suspension in a one milliliter syringe and pass it through a 70 micrometer cell strainer into a clean 50 milliliter centrifuge tube.
Finally, place the samples on ice. To freeze bone marrow for later use, centrifuge the cells at 1000 G for five minutes at room temperature. Discard the supernatant and resuspend the red pellet in two milliliters of antifreeze.
Then, incubate it at 80 degrees Celsius overnight. If frozen, defrost the suspension rapidly at 37 degrees Celsius. Transfer the cell suspension to a clean sterile tube containing three milliliters of DMEM/F-12 media.
Next, pellet the cells by centrifugation. Discard the supernatant and resuspend the pellet in three milliliters of the prepared DMEM/F-12 media. Count the cells and plate them in non-TC coated plastic ware in the prepared DMEM/F-12 media.
Next, incubate the cells at 37 degrees Celsius with 5%carbon dioxide. On the fifth day, feed the cells by adding 50%of the original volume of DMEM/F-12 media. On the sixth day, stimulate the cells by adding prepared GM-CSF to a final concentration of 50 nanograms per microliter directly to the cell culture media.
On day seven, remove all the media from the cells. Then, wash the cells briefly with pre-warmed PBS and add DMEM/F-12 media containing 2%low endotoxin FBS, 1X penicillin or streptomycin, L-glutamine, 25 nanograms per microliter M-CSF, and 50 nanograms per microliter GM-CSF. Finally, incubate the cells overnight in media to activate the macrophages to M0 phenotype.
For M1 phenotype, stimulate the cells with 100 nanograms per microliter LPS, and 10 nanograms per microliter IFN-gamma. For M2 phenotype, stimulate the cells with 100 nanograms per microliter IL-4. Incubate the cells overnight.
On day eight, cells are ready for harvest or downstream processing according to the researcher's needs. Media supplemented with 10%FBS had similar nitrite and tetrahydrobiopterin, regardless of M-CSF and GM-CSF, or LCM. But total biopterin levels were higher in LCM supplemented media.
However, more variability between different LCM batches was observed. PCR showed a 1030 base pair product in wild type mice, while GCH knockout mice produced a 1392 base pair product, confirming excision of the critical exons. Tamoxifen treatment abolished GCH protein in the knockout cells.
Knockout and control cells still produced iNOS, following LPS and IFN stimulation. BMDMs cultured from conditional GCH knockout mice exhibited significantly diminished tetrahydrobiopterin and decreased nitrite. The same cells cultured in LCM media had no GCH1 expression.
However, the cells produce significant amounts of tetrahydrobiopterin and nitrite following knockout. On the eighth day, there were no significant differences in morphology between nonactivated control and GCH knockout BMDMs at different concentrations of Tamoxifen. The BMDMs display the features expected of unstimulated macrophages, with round adherent cells and elongated extremities.
When working with primary cell like this, it's imperative to sterilize all tissue and equipment thoroughly. Furthermore, choosing the right media and supplement is important to maintain a low nitric oxide and BH4 environment. Cell pellets and media can be used for various downstream application, with a focus on the role of nitric oxide, biopterin, or cellular redox state.
It may include HPLC, qPCR, and Western blot, amongst other.
