Anti abca1

Single-cell suspensions from surgically resected CLM tissues from four patients were obtained as described in the previous paragraph. To assess expression of lipid transporters (ABCA1 and ABCG1), ApoE, and CD36, the following antibodies were included in the cocktail: anti-ABCA1 (Invitrogen; rabbit polyclonal), anti-ABCG1 (Novus Biologicals; rabbit polyclonal), anti-ApoE (BioLegend; clone E6D7), and anti-CD36 (BioLegend; clone 5–271). Detection of ABCG1 and ApoE was performed by intracellular staining with BD Cytofix/Cytoperm Solution (BD Biosciences). Cells were acquired on a FACSAria III (BD Biosciences) following the gating strategy shown in Fig. S2.
For lipid staining, cells were stained with the same cocktail used for cell sorting and then incubated with HCS LipidTOX Deep Red Neutral Lipid Stain (ThermoFisher; dilution 1:200) in PBS for 30 min at room temperature. Cells were immediately washed with PBS and acquired by flow cytometer. Lipid content of L-TAMs and S-TAMs was evaluated as mean fluorescence intensity (MFI) of the dye.
To test lipid uptake, cells were incubated with 0.8 µM of the fluorescent dye BODIPY 558/568 C12 (ThermoFisher) in PBS containing 0.1% BSA (fatty acid–free) for 1 min at 37°C. Cells were immediately washed with cold PBS containing 0.2% BSA and acquired by flow cytometer. Lipid uptake of L-TAMs and S-TAMs was evaluated as MFI of the dye.

Cells were treated with 0.5 mM of aspirin or 5 μg/ml of apoA-I for 12 h and then harvested with RIPA buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1% Triton-x100, 1% SDS, 0.5% Deoxycholate) containing the protease inhibitor cocktail (Sigma) after washing three times with DPBS. Samples were homogenized by a sonicator (Hielscher, UP50F) and passed through a 25-gauge needle. The suspension was clarified by centrifugation at 5,000 rpm for 10 min. The protein concentration of samples was determined by the Lowry method and was separated by SDS-PAGE and then transferred to a PVDF membrane (Roche). The membranes were incubated with anti-ABCA1 (1: 2,000; Invitrogen) and anti β-actin (1: 10,000, Sigma) overnight. Blots were washed with TBST and incubated with a horseradish peroxidase-conjugated secondary anti-mouse antibody. The membranes were developed with the ECL chemiluminescence system. The WB data was analyzed using ImageJ software and normalized with β–actin or GAPDH. All experiments were run in triplicate.