This work was supported partially by the University of Louisville start-up funds and National Cancer Institute (Bethesda, MD) grants R01CA177679, R01CA180986.

The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of University of Louisville.
1. Mouse Bone Marrow Derived Macrophages (BMDMs)
<ol>
	<li>Euthanize a 6- to 8-week-old, healthy wild type mouse using CO2. Pin it down to a foam board and spray it with 70% ethanol until it is soaked. Remove the tibia/femur bones with forceps and scissors. Put them in a petri dish with 5 mL 1x phosphate-buffered saline (PBS). Perform all procedures under sterile conditions.</li>
	<li>Cut open both ends of the tibia/femur bone with a scissor in a sterile tissue culture hood. Flush the bone with a 25 G needle with PBS + 2% fetal bovine serum (FBS) into a 15 mL tube.</li>
	<li>Centrifuge the bone marrow cells at 500 x g, 4 &#176;C for 5 min.</li>
	<li>Resuspend the cell pellet in 1 mL red blood cell lysis buffer to lyse red blood cells (RBC) for less than 1 min. Dilute immediately with 9 mL 1x PBS. Centrifuge again as in step 1.3. Decant the supernatants.</li>
	<li>Resuspend the cell pellet in 10 mL 1x PBS, filter the cell suspension through a sterile 40 &#956;m&#160;nylon mesh into another 15 mL tube to remove cell debris/clumps. Centrifuge again as in 1.3. Decant the supernatants.</li>
	<li>Resuspend the cell pellet in 15 mL Roswell Park Memorial Institute medium (RPMI) 1640 with 5% FBS and 10 &#956;g/mL gentamicin, plate the cells in a 100-mm tissue culture dish at 37 oC incubator for 60 min. Gently swirl the dish, take out the floating cells, and count them.</li>
	<li>Plate 6x106 cells in a 100-mm dish with 12 mL macrophage differentiating media (RPMI 1640 with 5% FBS and 10 &#956;g/mL gentamicin, mixed with 30% L929 conditioned media10) with 10 ng/mL recombinant mouse macrophage colony-stimulating factor (M-CSF) for 2 days.</li>
	<li>After culturing for 2 days in a 37 &#176;C incubator with 5% CO2, feed each dish with 6 mL fresh macrophage differentiating media with 10 ng/mL M-CSF.</li>
	<li>On day 5, take 8 mL of the old media out without disturbing the cells and add 10 mL fresh macrophage differentiating media with 10 ng/mL M-CSF.</li>
	<li>On day 7, harvest the bone marrow-derived macrophages (BMDMs) using a cell lifter. 
	NOTE: Attached macrophages can also be dissociated with 5 mL 1 mM EDTA in 1x PBS for 5 to 10 min after removing the float cells and washing plate twice with 5 mL 1x PBS. Centrifuge the cells as in step 1.3., resuspend them in fresh macrophage differentiating media, and count them with a hemocytometer.
	<ol>
		<li>Confirm the BMDMs&#39; phenotype by flow cytometry as described5. Prepare the derived mouse macrophages in a certain concentration for the following studies.</li>
	</ol>
	</li>
</ol>
2. BSA-fatty Acid Conjugate Preparation
<ol>
	<li>Prepare 2 mM BSA in sterile PBS. For example, weigh 6.65 g BSA, add 30 mL 1x PBS to dissolve, then add more PBS until 50 mL, scaling components to make 2 mM BSA in 1x PBS.</li>
	<li>Prepare 5 mM individual sodium salt of fatty acids in 2 mM BSA. Heat to 37 &#176;C or higher, if necessary, and sonicate the mixture of 2 mM BSA and sodium salt of fatty acids until a clear solution is obtained. 
	NOTE: Compared to unsaturated fatty acids, saturated fatty acids need a higher temperature and more sonication time for dissolving.</li>
	<li>Filter the fatty-acid-BSA solution through a 0.22 &#956;m filter, aliquot it into 1.5 mL sterile microcentrifuge tubes, and store them at 4 &#176;C for short term use or at -20 &#176;C for long term storage. No precipitation should be observed after storage at 4 &#176;C in the refrigerator.</li>
</ol>
3. Saturated Fatty Acids Induce Cell Death of Mouse Bone Marrow Derived Macrophages
<ol>
	<li>Plate the BMDMs in a 24-well tissue culture plate with 0.4 x 106/mL in macrophage differentiating media.</li>
	<li>Treat the cells with the 0.4 mM palmitic acid and stearic acid for 16 to 24 h at a 37 &#176;C incubator with 5% CO2. Use BSA as the negative control.</li>
	<li>Harvest the cells with a cell lifter after treatment and spin down the cells at 500 x g, 4 &#176;C for 5 min.</li>
	<li>Stain the cells with Annexin V-Alexa 488 and 7-aminoactinomycin D (7-AAD) in 100 &#956;L annexin V binding buffer for 15 min at room temperature.</li>
	<li>Dilute the sample with 200 &#181;L of annexin-binding buffer, mix gently, then keep the samples on ice after the incubation period.</li>
	<li>Analyze the stained cells as soon as possible by flow cytometry5. Annexin V-Alexa 488 is detected in FITC channel and 7-AAD is detected in PerCP/PerCP-Cy5.5 channel.</li>
</ol>
4. Intracellular Ceramide Staining
<ol>
	<li>After fatty acid treatment, harvest the cells as in step 3.3. Then fix the cells in 4% paraformaldehyde for 30 min.</li>
	<li>Wash the sample with 1 mL 1x PBS, spin down as step in 3.3, and decant the supernatant.</li>
	<li>Stain the cells with anti-ceramide primary antibody (1:200 dilution) in 100 &#956;L 1x permeabilization buffer for 30 min.</li>
	<li>Wash and spin down the cells again as in step 4.2.</li>
	<li>Stain the cells with anti-mouse IgM secondary antibody (1:500 dilution) in 100 &#956;L 1x permeabilization buffer for 30 min.</li>
	<li>Wash and spin down the cells again as in step 4.2. Add 250 &#956;L 1x PBS and analyze the stained cells by flow cytometry5. 
	NOTE: See the detailed reagent list in the Table of equipment and reagents.</li>
</ol>

<ol>
	<li>Martins de Lima, T., Cury-Boaventura, M. F., Giannocco, G., Nunes, M. T., Curi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+toxicity+of+fatty+acids+on+a+macrophage+cell+line+(J774).">Comparative toxicity of fatty acids on a macrophage cell line (J774).</a> Clin Sci (Lond). 111 (5), 307-317 (2006).</li><li>Simard, J. R., Zunszain, P. A., Hamilton, J. A., Curry, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Location+of+high+and+low+affinity+fatty+acid+binding+sites+on+human+serum+albumin+revealed+by+NMR+drug-competition+analysis.">Location of high and low affinity fatty acid binding sites on human serum albumin revealed by NMR drug-competition analysis.</a> J Mol Biol. 361 (2), 336-351 (2006).</li><li>Penn, A. H., Dubick, M. A., Torres Filho, I. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+Acid+Saturation+of+Albumin+Used+in+Resuscitation+Fluids+Modulates+Cell+Damage+in+Shock:+In+Vitro+Results+Using+a+Novel+Technique+to+Measure+Fatty+Acid+Binding+Capacity.">Fatty Acid Saturation of Albumin Used in Resuscitation Fluids Modulates Cell Damage in Shock: In Vitro Results Using a Novel Technique to Measure Fatty Acid Binding Capacity.</a> Shock. , (2017).</li><li>Vusse, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Albumin+as+fatty+acid+transporter.">Albumin as fatty acid transporter.</a> Drug Metab Pharmacokinet. 24 (4), 300-307 (2009).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+Fatty+Acid+Binding+Protein+Promotes+Saturated+Fatty+Acid-Induced+Macrophage+Cell+Death+through+Enhancing+Ceramide+Production.">Adipose Fatty Acid Binding Protein Promotes Saturated Fatty Acid-Induced Macrophage Cell Death through Enhancing Ceramide Production.</a> J Immunol. 198 (2), 798-807 (2017).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+Fatty+Acid+binding+protein+promotes+skin+inflammation+induced+by+high-fat+diet.">Epidermal Fatty Acid binding protein promotes skin inflammation induced by high-fat diet.</a> Immunity. 42 (5), 953-964 (2015).</li><li>Wen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid-induced+NLRP3-ASC+inflammasome+activation+interferes+with+insulin+signaling.">Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling.</a> Nat Immunol. 12 (5), 408-415 (2011).</li><li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fatty+acid-binding+protein+E-FABP+restricts+tumor+growth+by+promoting+IFN-beta+responses+in+tumor-associated+macrophages.">Fatty acid-binding protein E-FABP restricts tumor growth by promoting IFN-beta responses in tumor-associated macrophages.</a> Cancer Res. 74 (11), 2986-2998 (2014).</li><li>Ulloth, J. E., Casiano, C. A., De Leon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Palmitic+and+stearic+fatty+acids+induce+caspase-dependent+and+-independent+cell+death+in+nerve+growth+factor+differentiated+PC12+cells.">Palmitic and stearic fatty acids induce caspase-dependent and -independent cell death in nerve growth factor differentiated PC12 cells.</a> J Neurochem. 84 (4), 655-668 (2003).</li><li>Weischenfeldt, J., Porse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+Marrow-Derived+Macrophages+(BMM):+Isolation+and+Applications.">Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.</a> CSH Protoc. , (2008).</li><li>Dansen, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-affinity+binding+of+very-long-chain+fatty+acyl-CoA+esters+to+the+peroxisomal+non-specific+lipid-transfer+protein+(sterol+carrier+protein-2).">High-affinity binding of very-long-chain fatty acyl-CoA esters to the peroxisomal non-specific lipid-transfer protein (sterol carrier protein-2).</a> Biochem J. 339 (Pt 1), 193-199 (1999).</li><li>Ulloth, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+methyl-beta-cyclodextrin+toxicity+in+NGF-differentiated+PC12+cell+death.">Characterization of methyl-beta-cyclodextrin toxicity in NGF-differentiated PC12 cell death.</a> Neurotoxicology. 28 (3), 613-621 (2007).</li></ol>

The authors declare no financial conflicts of interest.

The proper preparation of fatty acid solution is of critical importance to study the biological function of fatty acids. The neutralization of fatty acid increases its solubility in aqueous solution. However, sodium salts of fatty acids, especially saturated fatty acids, are still of low solubility in water or PBS as we observed. One method is to use 95 - 100% ethanol to help dissolve fatty acid1. Using this method, higher toxicity to cells may be observed even at lower concentrations for less toxic unsaturated fatty acids. Another way to prepare fatty acid solution is to utilize methyl-&#946;-cyclodextrin as a delivery system so that fatty acids have optimal conditions, remaining in solution in their monomeric form9,11. Fatty acids prepared with such a method appeared to have normal function, but conducting the method is complicated. The impact of methyl-&#946;-cyclodextrin on cell growth is that high concentration could lead to cell death12. For saturated stearic and palmitic acids, a high temperature (60 oC) is used to help with solubilization9.
Since serum albumin is a major fatty acid-binding protein in extracellular fluid, which aids in transporting fatty acid in the lymphatic and vascular systems, it is favored as the natural carrier for the functional study of fatty acids. To avoid the low solubility of fatty acid salt in aqueous solution, sonication will greatly facilitate the conjugation of fatty acid with BSA, thus aiding in fatty acid solubilization. It takes much longer (hours) to solubilize the saturated fatty acids than the unsaturated fatty acids (30 min) with same amount of volume and concentration. Although heating and sonication are critical for BSA-fatty acid conjugation, a high temperature (less than 50 oC) is suggested for obtaining clear BSA-PA, BSA-SA conjugate solutions, and a low temperature is strongly recommended for solubilizing unsaturated fatty acids due to their susceptibility to oxidation at high temperature. The 5:2 molar ratio of fatty acid to BSA is considered good compared to the 3:1 or 6:1 molar ratio that was published7. This can ensure less totally-free fatty acid in the solution to avoid the toxicity of fatty acid soap. BSA-fatty acids prepared this way appear to be stable solutions (no precipitation, at least) at 4 &#176;C for at least 3 months as shown in their functionality on macrophage cell death. Seal the cap of the tube if prepared fatty acids are not to be used for a long period of time.
When a clear solution of BSA-fatty acid in PBS is obtained, consistent results from fatty acid treatment are more likely warranted. However, starving and well-fed macrophages respond quite differently to fatty acid treatment, especially saturated fatty acids. Macrophages with excess nutrients are more susceptible to saturated fatty acid-induced cell death than starving or starved ones because hungered cells can metabolically detoxify the saturated fatty acids due to their survival and growth request. Such cell death is highly associated with accumulated cellular ceramide levels. Of note, ceramide toxicity occurs only when intracellular ceramide levels reach a certain threshold. To ensure consistent results from fatty acid treated macrophages or any other type of cells, cells should be cultured with fresh media with plentiful nutrients. Any sub-optimal cell culture conditions could result in sub-optimal data.
Overall, these protocols provide much needed details for studying the impact of fatty acids on macrophages by other laboratories. Compared to other methods, this direct approach generates BSA-fatty acid conjugates without using an extra solvent like ethanol. The most challenging step is to prepare accurate, stable BSA-fatty acid solution because sonication generates heat and it is hard to control temperature. Fortunately, unsaturated fatty acids are easy to prepare in a short time and remain stable, while saturated fatty acids appear to stand relatively high temperatures because they are saturated and resistant to oxidation in such a condition. So far, we have not observed any issue or limitation with BSA-fatty acid conjugates prepared this way. Modification of our protocol would be necessary if more sensitive experiments are needed.

Fatty acids play a critical role in energy metabolism and in the synthesis of membrane phospholipids in different kinds of cells. Fatty acids have a low aqueous solubility. Appropriate preparation of fatty acid is of critical importance for studying the biological functions of fatty acids in mammalian cells. When fatty acids are prepared with ethanol, many fatty acids may show their toxic soap (detergent) effect on the cell membrane, even at relatively low concentrations1. As a natural, major transporter for free fatty acids in the serum, serum albumin is considered a good carrier for fatty acid delivery in vitro for fatty acid function assays2,3,4. However, the details of the preparation of fatty acid and serum albumin conjugate are usually not available even though many research papers using fatty acids have been published.
Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein5,6,7,8. They actively uptake saturated and unsaturated fatty acids which may regulate their immune functions. To study the impact of fatty acids on macrophages and other cells, different methods of fatty acid preparation were applied1,7,9. Using appropriately prepared fatty acid/serum albumin conjugates to investigate the impact of fatty acids on macrophage function is of critical importance in obtaining biologically meaningful data. Studies on the impact of fatty acids on macrophage function can provide basic knowledge and potential therapeutic targets relating to fatty acid metabolism in macrophage-involved diseases.

<table><tbody><tr><td>CPX Ultrasonic Bath&amp;nbsp;</td><td>Bransonic</td><td>Model 2800</td><td></td></tr><tr><td>Sodium palmitate (PA)</td><td>Nu-Chek Prep, Inc.</td><td>S-1109</td><td>M.W. 278</td></tr><tr><td>Sodium stearate (SA)</td><td>Nu-Chek Prep, Inc.</td><td>S-1111</td><td>M.W. 306</td></tr><tr><td>Bovine serum albumin (BSA), fatty acid-free</td><td>Fisher Scientific</td><td>9048-46-8</td><td></td></tr><tr><td>Mouse macrophage colony stimulating factor (mM-CSF)&amp;nbsp;</td><td>Cell Signaling Technology,&amp;nbsp; Inc.</td><td>5228</td><td></td></tr><tr><td>RPMI 1640</td><td>VWR International</td><td>71002-878</td><td></td></tr><tr><td>Annexin V, Alexa Fluor 488 conjugate</td><td>Fisher Scientific</td><td>A13201</td><td></td></tr><tr><td>7-AAD</td><td>BD Biosciences</td><td>559925</td><td></td></tr><tr><td>Monoclonal anti-ceramide antibody (mouse IgM)</td><td>Sigma</td><td>C8104-50TST</td><td>Clone: MID 15B4</td></tr><tr><td>Goat Anti-Mouse IgM Antibody, &amp;micro; chain, FITC conjugate</td><td>Sigma</td><td>AP128F</td><td></td></tr><tr><td>Fixation buffer</td><td>Biolegend</td><td>420801</td><td></td></tr><tr><td>Permeabilization buffer</td><td>Ebioscience</td><td>4307693</td><td></td></tr><tr><td>Red Blood Cell Lysis Buffer&amp;nbsp;</td><td>Sigma</td><td>11814389001</td><td></td></tr><tr><td>Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>L929 cells</td><td>ATCC</td><td>CCL-1</td><td></td></tr><tr><td>Corning&amp;nbsp;Cell Lifter&amp;nbsp;</td><td>Fisher Scientific</td><td>07-200-364</td><td></td></tr><tr><td>Note: M.W. is for molecular weight.</td><td></td><td></td><td></td></tr></tbody></table>

saturated fatty acids induce ceramide-associated macrophage cell death

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated fatty acids, which might play a critical role in regulating their immune functions. Numerous studies have shown that various fatty acids, saturated or unsaturated, may possess different impacts on cell growth and function. However, the approaches used for fatty acid preparation vary, which may lead to non-physiological results. Serum albumin, a natural carrier for fatty acids in mammalian peripheral blood, is recommended for forming a conjugate complex with the sodium salt of fatty acids to study fatty acid function in mammalian cells, thus minimizing the toxicity of fatty acid soap. Thus, a simple, relatively quick heating and sonicating method is developed and presented here for BSA-fatty acid conjugate formation. We describe a protocol using saturated fatty acids, especially stearic acids to induce severe cell death in mouse bone-marrow derived macrophages. We further demonstrate that saturated fatty acid-induced cell death is positively associated with accumulated cellular ceramide levels. This method can be extended for studies of the impact of fatty acid on other mammalian cells.

Obesity increases free fatty acid concentrations in serum. As professional phagocytes, macrophages actively take up fatty acids to maintain host homeostasis. During these processes, overloaded lipids may induce macrophage cell death. To this end, we cultured BMDMs in vitro with obese levels of dietary fatty acids and measured macrophage cell death using flow cytometric staining. Compared to the BSA control, saturated fatty acids, in particular stearic acids, induced significant cell death of BMDMs. Dead cells were shown as double positive populations stained by Annexin V and 7-AAD (Figure 1a-b). Of note, unsaturated fatty acids did not induce significant macrophage cell death5. The data suggest the toxic effect of increased levels of free saturated fatty acids in vivo.
Similarly, using a macrophage cell line whose viability was susceptible to the treatment of either palmitic acids or stearic acids5, we found that macrophages&#39; susceptibility to palmitic acid-induced cell death was also affected by the number of cells in the culture (Figure 2a). Simply put, under the same cultural conditions, the more macrophages in the wells, the less cell death induced by the fatty acids. Moreover, palmitic acid-induced cell death was positively associated with accumulated levels of cellular ceramides (Figure 2b). When macrophages were cultured in a high-density condition, medium nutrients were depleted faster for energy metabolism, these cells were less susceptible to palmitic acid-induced cell death compared to macrophages cultured in a low-density condition. In contrast, when cells were exposed to a nutrient-enriched condition (e.g., obesity), excess fatty acids can be metabolized to produce toxic ceramides, thereby leading to fatty acid-induced cell death. Thus, cell cultural conditions may bring variation to the results of saturated fatty acid-induced cell death.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56535/56535fig1.jpg" /> 
Figure 1: High concentration of fatty acids, especially stearic acids, induce macrophage cell death. BMDMs (day 7) were plated as 0.4 x 106/mL in fresh macrophage differentiating media. After cell attachment for 0.5 -1 h, BMDMs were treated with the designated concentration of each fatty acid for 24 h. Media alone and BSA were used as negative controls. After treatment, cells were lifted and harvested, then spun down at 500 g for 5 min at 4 &#176;C. Cells were stained with 7-AAD and Annexin V Alexa 488 in 100 &#956;L annexin-binding buffer for 15 min. Then add another 200 &#956;L annexin-binding buffer to each sample before flow cytometry analysis. (A) Demonstration of flow cytometry data of cells induced by fatty acids. (B) Summary of the cell death induced by fatty acids. Error bar represents sample SD. The cell death induced by palmitic acid (PA) and particularly stearic acid (SA) is statistically significant compared to BSA control&#39;s. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56535/56535fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56535/56535fig2.jpg" /> 
Figure 2: The availability of media nutrients impacts macrophages&#39; susceptibility to palmitic acid induced cell death. (A) Cell nutrition status impacts its tolerance to saturated fatty acid-induced stress. (B) Cell susceptibility to saturated fatty-acid induced cell death is highly correlated with accumulated cellular levels of ceramide. The media nutrient gradient was achieved by culturing the wild type macrophage cell line at 1M, 2M, 4M and 8M (M=1x106) in 6-mm petri dishes with 6 mL RPMI 1640 with 5% FBS and 10 &#956;g/mL gentamicin for 18 hr. The media color is more yellow due to more nutrient depletion when a greater number of cells are cultured in the dishes. More than 90% of the cells are still highly viable. The attached viable cells were lifted by pipetting after sitting in PBS for 5 min and re-cultured immediately at 0.2x106/mL in fresh media in 24-well plates. Cells were treated immediately with 0.4 mM palmitic acid for 18 h&#160;using BSA as the control for each source (nutrient gradient) of cells. Then all treated cells were harvested and stained with 7-AAD for cell death or stained with intracellular ceramide. Spontaneous cell death (7-AAD+) percentages were less than 8.6%. Treatment specific cell death was estimated as the % difference in 7-AAD+ between treatment and its own BSA control (a). The treatment specific ceramide level was estimated as the mean fluorescence intensity (MFI) difference between the treatment and its own BSA control (b). This experiment was repeated, and representative data are presented. Error bar represents sample SD. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56535/56535fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death at JoVE.com

Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

Macrophages highly express epidermal fatty acid-binding protein and adipose fatty acid-binding protein. They actively uptake saturated and unsaturated ...

saturated-fatty-acids-induce-ceramide-associated-macrophage-cell-death

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

9.2K Views.  University of Louisville. We illustrate a straight-forward method to derive murine primary macrophages from bone marrow cells and a simple method to prepare BSA-fatty acid conjugates. Then we demonstrate that saturated fatty acids can induce macrophage cell death, and such cell death is positively associated with cellular accumulation of ceramide levels.

Video: Saturated Fatty Acids Induce Ceramide-associated Macrophage Cell Death

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. Diet-induced obesity (DIO) is a valuable model in studying the role of macrophage heterogeneity; however, adequate macrophage isolations are difficult to acquire from inflamed tissues. In this protocol, we outline the isolation steps and necessary troubleshooting guidelines derived from our studies for obtaining a suitable population of tissue-resident macrophages from mice following 18 weeks of high-fat (HFD) or high-fat/high-cholesterol (HFHCD) diet intervention. This protocol focuses on three hallmark tissues studied in obesity and atherosclerosis including the liver, white adipose tissues (WAT), and the aorta. We highlight how dualistic usage of flow cytometry can achieve a new dimension of isolation and characterization of tissue-resident macrophages. A fundamental section of this protocol addresses the intricacies underlying tissue-specific enzymatic digestions and macrophage isolation, and subsequent cell-surface antibody staining for flow cytometric analysis. This protocol addresses existing complexities underlying fluorescent-activated cell sorting (FACS) and presents clarifications to these complexities so as to obtain broad range characterization from adequately sorted cell populations. Alternate enrichment methods are included for sorting cells, such as the dense liver, allowing for flexibility and time management when working with FACS. In brief, this protocol aids the researcher to evaluate macrophage heterogeneity from a multitude of inflamed tissues in a given study and provides insightful troubleshooting tips that have been successful for favorable cellular isolation and characterization of immune cells in DIO-mediated inflammation.

This protocol allows a researcher to isolate and characterize tissue-resident macrophages in various hallmark inflamed tissues extracted from diet-induced models of metabolic disorders.

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Biology

Detection of Inflammasome Activation and Pyroptotic Cell Death in Murine Bone Marrow-derived Macrophages

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote inflammatory and autoinflammatory diseases. Inflammasomes are activated by cytosolic pattern recognition receptors, including members of the NOD-like receptor (NLR) family. These receptors oligomerize upon the detection of microbial or damage-associated stimuli. Subsequent recruitment of the adaptor protein ASC forms a microscopically visible inflammasome complex, which activates caspase-1 through proximity-induced auto-activation. Following the activation, caspase-1 cleaves pro-IL-1&#946; and pro-IL-18, leading to the activation and secretion of these pro-inflammatory cytokines. Caspase-1 also mediates the inflammatory form of cell death termed pyroptosis, which features the loss of membrane integrity and cell lysis. Caspase-1 cleaves gasdermin D, releasing the N-terminal fragment which forms plasma membrane pores, leading to osmotic lysis.
In vitro, the activation of caspase-1 can be determined by labeling bone marrow-derived macrophages with the caspase-1 activity probe FAM-YVAD-FMK and by labeling the cells with antibodies against the adaptor protein ASC. This technique allows the identification of inflammasome formation and caspase-1 activation in individual cells using fluorescence microscopy. Pyroptotic cell death can be detected by measuring the release of cytosolic lactate dehydrogenase into the medium. This procedure is simple, cost effective and performed in a 96-well plate format, allowing adaptation for screening. In this manuscript, we show that activation of the NLRP3 inflammasome by nigericin leads to the co-localization of the adaptor protein ASC and active caspase-1, leading to pyroptosis.

We describe the detection of NLRP3 inflammasome activation on cellular basis using fluorescence microscopy and staining for active caspase-1 and the adaptor, ASC. A lactate dehydrogenase release assay is presented to detect pyroptotic lysis on a population basis. These techniques can be adapted to study many aspects of inflammasome biology.

Inflammasomes are innate immune signaling platforms that are required for the successful control of many pathogenic organisms, but also promote ...

Experimental Analysis of Apoptotic Thymocyte Engulfment by Macrophages

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution of inflammation. Accumulation of apoptotic debris in the body may trigger chronic inflammatory responses that lead to systemic autoimmune diseases over time. Impaired apoptotic cell clearance has been implicated in a variety of autoimmune diseases. Apoptotic clearance is a complex process rarely detected under physiological conditions. It involves abundant surface receptors and signaling molecules. Studying the process of apoptotic cell clearance provides insightful molecular mechanisms and subsequent biological responses, which may lead to the development of new therapeutics. Here, we describe protocols for the induction of apoptotic thymocytes, the preparation of peritoneal macrophages, and the analysis of apoptotic cell clearance by flow cytometry and microscopy. All cells will undergo apoptosis at a certain stage, and many residential and circulating cells can uptake apoptotic debris. Therefore, the protocol described here can be used in many applications to characterize apoptotic cell binding and ingestion by many other cell types.

Here, we present a protocol to prepare apoptotic thymocytes and peritoneal macrophages and analyze the efficiency of efferocytosis and the specific inhibitor-mediated blocking of apoptotic thymocytes engulfment. This protocol has a broad application in cell-mediated clearance of other particles including artificial beads and bacteria.

Cell apoptosis is a natural process and plays a critical role in embryonic development, homeostatic regulation, immune tolerance induction, and resolution ...

Inducing and Characterizing Vesicular Steatosis in Differentiated HepaRG Cells

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive fat accumulation leads to non-alcoholic fatty liver disease (NAFLD), &#160;which is potentially reversible and may evolve into non-alcoholic steatohepatitis (NASH) and eventually cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms linking lipid accumulation in hepatocytes with the progression to NASH, irreversible liver damage, fibrosis, cirrhosis, and even HCC still remains unclear. To this end, several in vitro and in vivo models have been developed to elucidate the pathological processes that cause NAFLD. In the present study, we describe a cellular model for the induction of liver vesicular steatosis that consists of DMSO-differentiated human hepatic HepaRG cells treated with the fatty acid salt sodium oleate. Indeed, sodium oleate-treated HepaRG cells accumulate lipid droplets in the cytoplasm and show typical features of steatosis. This in vitro human model represents a valuable alternative to in vivo mice models as well as to the primary human hepatocytes. We also present a comparison of several methods for the quantification and evaluation of fat accumulation in HepaRG cells, including Oil Red O staining, cytofluorimetric Bodipy measurement, metabolic gene expression analysis by qPCR, and coherent anti-Stokes Raman scattering (CARS) microscopy. CARS imaging combines the chemical specificity of Raman spectroscopy, a chemical analysis technique well-known in materials science applications, with the benefits of high-speed, high-resolution non-linear optical microscopies to allow precise quantification of lipid accumulation and lipid droplet dynamics. The establishment of an efficient in vitro model for the induction of vesicular steatosis, alongside an accurate method for the quantification and characterization of lipid accumulation, could lead to the development of early stage diagnosis of NAFLD via the identification of molecular markers, and to the generation of new treatment strategies.

In this study, we describe a detailed protocol for inducing liver vesicular steatosis in differentiated HepaRG cells with the fatty acid salt sodium oleate and employ methods for detection and quantification of lipid accumulation, including coherent anti-Stokes Raman scattering (CARS) microscopy, cytofluorimetric analysis, Oil red O staining, and qPCR.&#160;

Hepatic steatosis represents a metabolic dysfunction that results from an accumulation of triglyceride-containing lipid droplets in hepatocytes. Excessive ...

Medicine

Identification of Intracellular Signaling Events Induced in Viable Cells by Interaction with Neighboring Cells Undergoing Apoptotic Cell Death

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent live cells. Vital activities, such as survival, proliferation, growth, and differentiation, are among the many cellular functions modulated by apoptotic cells. The ability to recognize and respond to apoptotic cells appears to be a universal feature of all cells, regardless of lineage or organ of origination. However, the diversity and complexity of the response to apoptotic cells mandates that great care be taken in dissecting the signaling events and pathways responsible for any particular outcome. In particular, one must distinguish among the multiple mechanisms by which apoptotic cells can influence intracellular signaling pathways within viable responder cells, including: receptor-mediated recognition of the apoptotic cell, release of soluble mediators by the apoptotic cell, and/or engagement of the phagocytic machinery. Here, we provide a protocol for identifying intracellular signaling events that are induced in viable responder cells following their exposure to apoptotic cells. A major advantage of the protocol lies in the attention it pays to dissection of the mechanism by which apoptotic cells modulate signaling events within responding cells. While the protocol is specific for a conditionally immortalized mouse kidney proximal tubular cell line (BU.MPT cells), it is easily adapted to cell lines that are non-epithelial in origin and/or derived from organs other than the kidney. The use of dead cells as a stimulus introduces several unique factors that can hinder the detection of intracellular signaling events. These problems, as well as strategies to minimize or circumvent them, are discussed within the protocol. Application of this protocol should aid our expanding knowledge of the broad influence that dead or dying cells exert on their live neighbors, both in health and in disease.

Here, we present a protocol for the determination of intracellular signaling events induced in viable cells by physical interaction with adjacent dead or dying cells. The protocol focuses on signaling events induced by receptor-mediated recognition of the dead cells, as opposed to their phagocytic uptake or release of soluble mediators.

Cells dying by apoptosis, also referred to as regulated cell death, acquire multiple new activities that enable them to influence the function of adjacent ...

Live Cell Imaging of Lipid Droplet Size and Fusion Using Oil Red O and BODIPY

Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety of metabolic diseases, such as obesity, fatty liver disease, and diabetes. In hepatic cells, the sizes and numbers of LDs are signs of fatty liver disease. Moreover, the oxidative stress reaction, cell autophagy, and apoptosis are often accompanied by changes in the sizes and numbers of LDs. As a result, the dimensions and quantity of LDs are the basis of the current research regarding the mechanism of LD biogenesis. Here, in fatty acid-induced bovine hepatic cells, we describe how to use oil red O to stain LDs and to investigate the sizes and numbers of LDs. The size distribution of LDs is statistically analyzed. The process of small LDs fusing into large LDs is also observed by a live cell imaging system. The current work provides a way to directly observe the size change trend of LDs under different physiological conditions.

Author Spotlight: Evaluation of Lipid Droplet Size and Fusion in Bovine Hepatic Cells  

The present protocol describes how to use oil red O to dye lipid droplets (LDs), calculate the size and number of LDs in a fatty acid-induced fatty hepatocyte model, and use BODIPY 493/503 to observe the process of small LDs fusing into large LDs by live cell imaging.

Lipid droplets (LDs) are organelles that play an important role in lipid metabolism and neutral lipid storage in cells. They are associated with a variety ...

Analyzing LPS/ATP-Triggered Cell Death Mechanisms in THP-1 Cells

LPS and ATP-induced Death of PMA-differentiated THP-1 Macrophages and its Validation

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show&#160;that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.

Author Spotlight: THP-1 Macrophage Response to LPS/ATP &#8212; Unveiling the Pyroptosis, Apoptosis, and Necroptosis Spectrum

This protocol is based on LPS and ATP-induced death of PMA-differentiated THP-1 macrophages. We use flow cytometry to analyze Annexin V and 7-AAD double staining to detect cell death, using the whole cell and employing scanning electron microscopy to observe the cell membrane morphology.

Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced ...

Visualization of Caspase Activation in Human Monocyte-Derived Macrophages

Source: Bol&#237;var, B. E., et. al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/63162">Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages.</a> J. Vis. Exp. (2022).
This video demonstrates the activation of caspase proteins in human macrophages using bimolecular fluorescence complementation reporter proteins. The pro-domains of caspase-1 are fused with non-fluorescent fragments of Venus proteins, which are recruited to inflammasome complexes. The proximity of inflammasomes induces the refolding and fluorescence of Venus fragments, confirming caspase activation in the macrophages.

Wizualizacja aktywacji kaspazy w ludzkich makrofagach pochodzących z monocytów

Source: Bol&#237;var, B. E., et. al., Visualization of Inflammatory Caspases Induced Proximity in Human Monocyte-Derived Macrophages. J. Vis. Exp. 
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JoVE Encyclopedia of Experiments

Immunology

Immune Response

Immune Modulation

Assessment of the Activation of Multiple Caspases in Macrophages

Source: Han, J. et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64308">Evaluation of Caspase Activation to Assess Innate Immune Cell Death.</a>&#160;J. Vis. Exp.&#160;(2023)
This video demonstrates the assessment of multiple caspase activations in bone marrow-derived macrophages through stimulation with the influenza virus. Following macrophage lysis, heat-inactivation of lysate, and centrifugation, intracellular caspases are collected for subsequent immunological analysis.