Evaluation of foam cell formation in cultured macrophages: an improved method with Oil Red O staining and DiI-oxLDL uptake

Abstract

Macrophage-derived foam cell formation elicited by oxidized low-density lipoprotein (oxLDL) is the hallmark of early atherogenesis. Detection of foam cell formation is conventionally practiced by Oil Red O (ORO) staining of lipid-laden macrophages. Other methods include 1,1'-dioctadecyl-3,3,3'3'-tetra-methylindocyanide percholorate (DiI)-labeled oxLDL (DiI-oxLDL) uptake and Nile Red staining. The purpose of the present study is to report an optimized method for assessing foam cell formation in cultured macrophages by ORO staining and DiI-oxLDL uptake. After incubation with oxLDL (50 μg/ml) for 24 h, the macrophages were fixed, stained with ORO for just 1 min, pronounced lipid droplets were clearly observed in more than 90% of the macrophages. To test the in vivo applicability of this method, lesions (or foam cells) of cryosections of aortic sinus or primary mouse peritoneal macrophages from ApoE deficient mice fed a high cholesterol diet were successfully stained. In another set of experiments, treatment of macrophages with DiI-oxLDL (10 μg/ml) for 4 h resulted in significant increase in oxLDL uptake in macrophages as demonstrated by confocol microscopy and flow cytometry. We conclude that the optimized ORO staining and fluorescent labeled oxLDL uptake techniques are very useful for assessing intracellular lipid accumulation in macrophages that are simpler and more rapid than currently used methods.

Using rifapentine – hen egg lipoprotein conjugate as macrophage-targeted drug delivery carrier against intracellularStaphylococcus aureus

“…These results indicated that the RPT-heLDL conjugate had entered the cells successfully. The scavenger receptor-mediated uptake of chemically modified lipoproteins by macrophages, including oxidized and acetylated LDL, was found to be concentration dependent ( Xu et al, 2010). Our oil red O-staining results revealed a similar pattern to that of the uptake of RPT-heLDL by U937 macrophage, which also correlated well with the concentration.…”

“…human THP-1, human monocyte-derived macrophages and murine macrophage cell lines) (Scholz et al, 2004;Moore & Freeman, 2006;Xu et al, 2010). The results of the current study revealed that the FITClabeled protein successfully entered the cell and that oil red O-stained lipid droplets were well distributed throughout the cytosol of the U937 macrophages.…”

Loss of Apoptosis Regulator through Modulating IAP Expression (ARIA) Protects Blood Vessels from Atherosclerosis

“…At the time of protein extraction, both cells transfected with ARIA-FLAG or empty vector were nearly confluent, and no significant difference of confluency was detected between the groups. Murine peritoneal macrophages (PMs) were prepared as described previously (22). Briefly, thioglycolate (2 ml of 3% solution in water) was intraperitoneally injected in age-and sex-matched WT and ARIA-deficient mice.…”

“…Foam Cell Formation-Foam cell formation was performed as described previously (22,23). Briefly, macrophages were cultured on chamber slides at a density of 5.…”

Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis

“…Cells that have been plated on cover glassbottom dishes or cytospun can be analyzed for shape to help determine their phenotype, though this can also be per formed on an imaging flow cytometer (Majka et al, 2012). Sorted cells can also be used for in vitro functional assays like antigen presentation (Choi et al, 2009), cytokine production by cytometric bead array (Morgan et al, 2004), phagocytosis (Xu et al, 2010), efferocytosis of apoptotic cells (Friggeri et al, 2010), and killing of pathogens (Drevets et al, 2015).…”