The overall goal of this experiment, in which macrophages are treated with saturated fatty acids, is to demonstrate that saturated fatty acid-induced macrophages cell death is associated with accumulated cellular levels of Ceramides. This method can help answer key questions in the field of metabolic inflammation. Such as:saturated fatty acid-induced cell death and inflammation.
The main advantage of this technique is to provide a simple but detailed approach to propel BSA fatty acid conjugates. Generally, individuals new to this method will struggle because it is challenging to prepare accurate stable BSA fatty acid conjugate solution with consistent results. Begin this procedure with removal of the tibia and femur bones from a euthanized six to eight week old healthy wild-type mouse, as described in the text protocol.
Cut open both ends of the tibia and femur bones with a scissor and a sterile Tissue Culture Hood. Flush the bones with a 25 gauge needle with PBS plus two percent Fetal Bovine Serum into a 15 milliliter tube. Centrifuge the bone marrow cells at 500 g, four degrees celsius for five minutes.
Next, re-suspend the cell pellet in one-milliliter of Red Blood Cell Lysis buffer to lyse red blood cells for less than one minute. Dilute the cells immediately with nine milliliters of 1x PBS. Centrifuge again, as before, and decant the supernatant.
Re-suspend the cell pellet in 10 milliliters of 1x PBS. Then, filter the cell suspension through sterile 40 micron Nylon Mesh into another 15 milliliter tube to remove cell debris and clumps. Following centrifugation as before, re-suspend the cell pellet in 15 milliliters of Roswell Park Memorial Institute medium 1640 with five percent FBS and 10 micrograms per milliliter of Gentamicin.
Then, plate the cells in a 100 millimeter Tissue Culture Dish and put it in a 37 degree Celsius incubator for 60 minutes. Gently swirl the dish, take out the floating cells, and count them. Next, plate 6, 000, 000 cells in a 100 millimeter dish with 12 milliliters of Macrophage Differentiating Media and 10 nanograms per milliliter of recombinant Mouse Macrophage Colony Stimulating Factor, or MCSF, for two days.
After culturing for two days in a 37 degrees Celsius incubator with five percent CO2, feed each dish with six milliliters of fresh Macrophage Differentiating Media containing 10 nanograms per milliliter of MCSF. On day five, take eight milliliters of the old media out without disturbing the cells and add 10 milliters of fresh Macrophage Differentiating Media with 10 nanograms per milliliter of MCSF. On Day seven, harvest the Bone Marrow Derived Macrophages, or BMDMs, using a cell lifter.
Confirm the BMDMs'phenotype by Flow Cytometry, as referenced in the text protocol. To prepare the BSA Fatty Acid Conjugate, prepare five millimolar individual Sodium Salt of Fatty Acids in two millimolar BSA. Heat the mixture to 37 degrees Celsius or higher if necessary.
Then, Sonic Heat the mixture of two millimolar BSA and Sodium Salt of Fatty Acids until a clear solution is obtained. Following sonication, filter the fatty acid BSA solution, through a 0.22 micron filter. Then, aliquot the filtered solution into 1.5 milliliter sterile microcentrifuge tubes.
Store the tubes at four degrees Celsius for short term use, or at minus 20 degrees Celsius for long term storage. No precipitation should be observed after storage at four degree Celsius in the refrigerator. Plate the BMDMs in a 24-well tissue culture plate with 0.4 million cells per milliliter in macrophage differentiating media.
Treat the cells with the 0.4 millimolar Palmitic acid and Stearic acid for 16 to 24 hours in a 37 degree Celsius incubator with five percent CO2. Use BSA as the negative control. Harvest the cells with a cell lifter after treatment.
Then, spin down the cells at 500 g, four degrees Celsius for five minutes. Stain the cells with Annexin V-Alexa 488 and 7-AAD in 100 microliters of Annexin-V binding buffer for 15 minutes at room temperature. Dilute the sample with 200 microliters of Annexin binding buffer and mix gently.
Keep the samples on ice after the incubation period. Analyze the stained cells as soon as possible by Flow Cytometry. Annexin V-Alexa 488 is detected in the FITC channel and 7-ADD is detected in the PerCP PerCP-Cy5.5 channel.
After fatty acid treatment, harvest the cells as before. Then, fix the cells in four percent Paraformaldehyde for 30 minutes. Wash the sample with one-milliter of 1x PBS before spinning down the sample as before.
After decanting the supernatant, stain the cells with Anti-Ceramide Primary Antibody in 100 microliters of 1x Permeabilization Buffer for 30 minutes. After washing and centrifuging the cells as before, stain the cells with Anti-Mouse IgM Secondary Antibody in 100 microliters of 1x Permeabilization Buffer for 30 minutes. Wash and spin down the cells again as before.
Then, add 250 microliters of 1x PBS and analyze the stained cells by Flow Cytometry. BMDMs were cultured in-vitro with obese levels of dietary fatty acids and macrophage cell death was measured using Flow Cytometric staining. Compared to the BSA control, saturated fatty acids, in particular Steric acids, induced significant cell death of BMDMs.
Dead cells were shown as double positive population stained by Annexin V and 7-AAD. Moreover, saturated fatty acid-induced cell death is positively associated with accumulated cellular Ceramide levels in macrophages. After watching this video, you should have a good understanding of how to prepare BSA Fatty Acid Conjugations and to treat macrophages with BSA Fatty Acid Conjugates to study cell death and inflammation.
On its development, this technique paved the way for researchers in the field of metabolic inflammation to explore saturated fatty acid-induced cell death and inflammation in obesity.
