The use of different energy substrates is both cell type specific and disease indicative. Measuring the consumption of the specific substrates can shed light on both normal biology and pathology. The technique does not need specialized equipment and is easy to scale.
It's also easier to use than previous published methods. Understanding changes in substrate oxidation rate will allow the development of dietary and pharmacological interventions for metabolic disorders. Although demonstrated with bone tissue this technique is easily customized to study metabolic flexibility in all cell types providing an understanding of both the causes and consequences of metabolic shifts.
After sacrificing the pups of three to five postnatal days, transfer them to ice cold D PBS containing penicillin streptomycin dual antibiotic solution. Expose the calvaria by removing the skin and soft tissue. Collect the middle region by cutting the surrounding tissues from back to front in D PBS.
Scratch the inner and outer surfaces of the calvaria gently using tweezers to help release the cells upon subsequent digestion. Prepare 2 and 4 milligrams per milliliter solutions of collagenase type two in D PBS and filter each solution with a fresh 0.22 micrometer filter. First, digest the cleaned calvaria with two milligrams per milliliter collagenase solution for 15 minutes, and then discard the digestion solution.
Next, digest the clean samples in four milligrams per milliliter collagenase solution thrice for 15 minutes each time. Then, pull the digestion solution and save it. Filter the digestion solution through 70 micrometer cell strainers and centrifuge the filtrate at 300 RCF for five minutes.
Re suspend the cell pellet in complete MEM alpha medium containing 10%FBS and penicillin streptomycin dual antibiotic solution. Count and seed the cells in a 10 centimeter plate. Culture the cells in a 37 degree Celsius incubator with 5%carbon dioxide for three days.
Then, dissociate the cells at 37 degrees Celsius with 0.25 Trypsin EDTA. After digestion, count and seed the cells in a 24 well cell culture plate with a complete MEM alpha medium containing 10%FBS and penicillin streptomycin dual antibiotic solution. Seed at least four wells per cell type per substrate, and at least three extra wells for each cell type for pre assay counting.
Culture the cells at 37 degrees Celsius overnight. After removing the muscle and connective tissue from eight week old mice and collecting femurs and tibias, cut and discard both ends of the bones with sharp scissors. Flush out the bone marrow from one femur and one tibia into a fresh 10 centimeter Petri dish with a syringe fitted with a 23 gauge needle containing 15 milliliters of complete MEM alpha medium with 10%FBS penicillin streptomycin dual antibiotic solution, and 10%CGM 14 12 conditioned medium.
Culture the cells in the Petri dish in a 37 degree Celsius incubator with 5%carbon dioxide. After three days discard the culture media and rinse the cells with D PBS. Dissociate the attached cells at 37 degrees Celsius with 0.25%Trypsin EDTA for five minutes.
After centrifugation, re suspend the cell pellet in the BMM medium. Count and seed the cells in a 24 well cell culture plate per the procedure described previously. Culture at 37 degrees Celsius overnight in the BMM medium before starting the assays.
Wash the cells in the extra wells twice with D PBS. Dissociate the cells with 0.25%Trypsin EDTA. Re suspend 20 microliters of digested cells in D PBS.
With 20 microliters of acridine orange and propidium iodide dye solution, determine the number of live cells with an automated cell counter and record the number. Wash the cells in the assay wells twice with D PBS. Add 500 microliters of hot medium to each assay well in the RAM designated tissue culture hood.
After sealing the plate with parafilm, incubate the cells in an RAM designated incubator at 37 degrees Celsius for 4 hours. During incubation, cut filter paper into circular pieces, slightly bigger than the area inside the cap of the 1.5 milliliter micro centrifuge tube and insert the paper snugly into the cap. Add 200 microliters of one 1 molar Perchloric acid to each tube and 20 microliters of sodium hydroxide to the filter paper fitted inside the cap.
After incubating the cells, transfer 400 microliters of culture medium from each well to the prepared tubes and close the caps immediately. Leave the tubes in a tube rack at room temperature for one hour. Parallelae during incubation set up a scintillation vial for each tube and fill it with four milliliters of scintillation fluid.
Transfer each piece of filter paper to a scintillation vial and incubate at room temperature for 30 minutes. Perform wipe tests on the tissue culture hood, the water bath, refrigerator, incubator, sink, ground, and any other working area for potential RAM contamination. Put the paper wipes into scintillation vials containing scintillation fluid.
Measure carbon 14 radio activity in the scintillation vials with a scintillation counter and record the reading results. Decontaminate the working environment, according to radiation safety guidelines, if necessary. The figure compares substrate oxidation by primary calvaria pre osteoblasts versus BMMs.
After the primary cells are passaged and cultured in complete MEM alpha medium overnight, they typically reach 80 to 90%confluency and exhibit their characteristic morphology. The calvarial pre osteoblasts are notably larger than BMMs. The results of substrate oxidation showed that the oxidation rate for each substrate is significantly higher in calvarial pre osteoblasts than BMMs, likely indicating higher energy production by oxidative phosphorylation in the pre osteoblasts.
The inserted third paper should be slightly bigger than a 1.7 milliliter ebin top tube cap. This method can be used in conjunction with oxygen consumption and to determine the overall rate of oxidative phosphorylation and lactative production in the cells. This protocol can be used to determine substrate usage during distinct differentiation stages or disease settings.
With this knowledge we can develop interventions for metabolic disorders.
