Pacific orange conjugated anti cd14 antibody

Frozen cell suspensions were thawed and stained using a combination of monoclonal antibodies as previously described.19, 20 A biotinylated anti–CADM1 antibody (clone 3E1) was purchased from MBL (Nagoya, Japan). A Pacific Orange‐conjugated anti–CD14 antibody was purchased from Life Technologies (Carlsbad, CA, USA). All other antibodies (anti–CD3, anti–CD4 and anti–CD7) and streptavidin‐PE were obtained from BioLegend (San Diego, CA, USA). A FACSAria (BD Immunocytometry Systems, San Jose, CA, USA) was used for the flow cytometric analysis. Acquired raw data were analyzed using FlowJo software (TreeStar, San Carlos, CA, USA). The gating procedure used here was the same as that described previously.19 Briefly, PI⁺ cells were gated out, and then CD14⁺ cells were gated out. Next, a CADM1 versus CD7 plot was constructed for CD4⁺ cells. As in our previous reports, all analyzed cases were categorized into four groups based on the proportion of CADM1⁺CD7^dim (D) and CADM1⁺CD7⁻ (N) cells: G1 (D + N ≤ 10%), G2 (10% < D + N ≤ 25%), G3 (25% < D + N ≤ 50%) and G4 (50% < D + N).20

Peripheral blood mononuclear cells were isolated from whole blood by density gradient centrifugation, as described previously.8 (link) An unlabeled CADM1 antibody (clone 3E1) and an isotype control chicken IgY antibody were purchased from MBL (Nagoya, Japan). These were biotinylated (by primary amine biotinylation) using biotin N-hydroxysuccinimide ester (Sigma Aldrich, St. Louis, MO, USA). A Pacific Orange-conjugated anti-CD14 antibody was purchased from Life Technologies (Carlsbad, CA, USA). All other antibodies were obtained from BioLegend (San Diego, CA, USA). Cells were stained using a combination of biotin-CADM1, allophycocyanin (APC)-CD7, APC-Cy7-CD3, Pacific Blue-CD4 and Pacific Orange-CD14. After washing, phycoerythrin (PE)-conjugated streptavidin was applied. Propidium iodide (PI [Sigma Aldrich]) was added to the samples to stain dead cells immediately before flow cytometry. A FACSAria instrument (BD Immunocytometry Systems, San Jose, CA, USA) was used for all multicolor flow cytometry and fluorescence-activated cell sorting (FACS). Data were analyzed using the FlowJo software (TreeStar, San Carlos, CA, USA). The gating procedure was as described previously.10 (link) Briefly, PI⁺ cells and then CD14⁺ cells were gated out. Next, a CADM1 versus CD7 plot for CD4⁺ cells was constructed (Fig.1a).