Naphthol as d chloroacetate esterase

1.07 × 10⁵ OP9 cells were plated in a 12-well plate the day before sorting. The low-density PBMC fraction was isolated by centrifugation over a Ficoll-Paque Plus density gradient (GE Healthcare Life Sciences). The PBMC layer was collected, and cells were washed and stained with CD14-APC-Cy7, HLA-DR-APC, CD15-PE, and Aqua Live/Dead Fixable 405. After sorting, MEMα without nucleosides was removed from the OP9 cells wells and replaced with IMDM (Thermo Fisher Scientific) supplemented with 20% FBS and 1% penicillin–streptomycin. Up to 3.5 × 10⁵ sorted cells were added per well in a total volume of 3 ml of IMDM in the presence of 100 ng/ml of G-CSF and 25 ng/ml of GM-CSF (Peprotech) or 100 ng/ml of M-CSF. At day 3, 2 ml of fresh media supplemented with fresh cytokines were added on the top. At day 4, cells were harvested by pipetting up and down. Human CD45⁺ cells were separated from murine OP9 cells by magnetic separation using biotinylated anti-human CD45 antibody (Miltenyi Biotec), streptavidin-coated microbeads (Miltenyi Biotec), and magnetic-activated cell sorting columns (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then stained with the May-Grünwald-Giemsa kit or the naphthol AS-D chloroacetate esterase (Sigma-Aldrich) kit or stained for flow cytometry.

Mouse tissues were fixed in 4% paraformaldehyde overnight, and embedded in paraffin. The TMA was created by taking two 0.8-mm punches from 191 independent donor human synovial sarcoma tumor blocks from pathology archives and arraying them into recipient blocks using a Beecher MTA-1 manual TMA apparatus as previously described (Lahat et al., 2010 (link)). Paraffin-embedded tissues were stained by immunohistochemistry by rehydrating slides through a citrosolv and ethanol dilution wash. Antigen-retrieval was performed in 10 mM sodium citrate (pH 6.0). Sections were immunostained with primary antibodies followed by horseradish peroxidase detection methods and counterstained with hematoxylin. The complete list of antibodies can be found in Table S7. Leder staining with Naphthol AS-D chloroacetate esterase (Sigma-Aldrich) was performed per manufacturer recommendations.

MPO levels in the spleen, as an assessment of neutrophil influx in tissue homogenates, were measured 24 h post-CLP by ELISA according to the manufacturer’s instructions (#EMMPO; Thermo Fisher Scientific, Waltham, Mass). Similar to cytokine analysis, the 24 h time point was selected as myeloid cells are known to respond robustly and rapidly to septic stimuli within this time frame. To measure esterase levels, spleen sections were stained with naphthol AS-D chloroacetate esterase (Sigma-Aldrich, St. Louis, Mo) according to the manufacturer’s instructions. The images were collected with the Nikon Eclipse 80i microscope using a ×20 and ×40 objective and Spot RT3 camera. Slides were randomly screened and blindly evaluated with three to six images acquired per specimen (42 (link)–44 (link)).