Anti cd45 apc clone 2d1

Fresh tissues (>0.5 g) for single-cell isolation were obtained directly after surgery and stored in 10% FCS at 4°C for a maximum of 16 hr. Normal adjacent tissues were obtained >10 cm from the bulk tumour but processing often failed due to the rigidity of normal tissue samples. Excess blood was removed by a PBS wash. Tissues were dispersed into ~4 mm pieces using razor blades. Further dissociation of cells was enzymatically obtained using collagenase I (0.2%) and dispase (2.5 U/mL) in DMEM with 2% FCS, accompanied by actinomycin D (74 μg/mL), DNAse (25 KU), and calcium chloride dehydrate (5 mM). The solution was incubated in a T25 filter flask for 1 hr at 37°C while shaking. After incubation, cells were sieved through a 100 μm nylon filter and spun down. Red blood cells were lysed in shock medium for 10 min on ice. After two washes, cells were stained for 30 min with anti-PECAM1-PE (clone WM59, ABD Serotec, Kidlington, UK), anti-CD45-APC (clone 2D1, BD, San Jose, CA, USA) and anti-EpCAM-FITC (clone 9c4, Biolegend, San Diego, CA, USA). Cells were washed, filtered (100 μm) and subjected to fluorescence-activated cell sorting (FACS Aria, BD) using a 100 μm nozzle (25 psi). Various cell populations are indicated in Supplementary Figure 1. Collected cells were spun down and immediately dissolved into 500 μL TRIzol (ThermoFisher Scientific, Waltham, MA, USA).

Peripheral whole blood samples were collected in sterile BD Vacutainer^® EDTA (ethylenediamine-tetraacetic acid) blood collection tubes (BD Biosciences). The samples were incubated in BD Trucount™ tubes (BD Biosciences) with the following monoclonal antibodies to identify lymphocyte sub-populations (BD Biosciences): anti-CD45-PerCP (clone 2D1), anti-CD3-FITC (clone UCHT-1), anti-CD4-APC (clone RPA-T4), anti-CD8-PE (clone RPA-T8), anti-CD16-PE (clone 3G8), anti-CD19-APC (clone HIB19), anti-CD56-PE (clone NCAM16.2). The following antibodies were used for monocyte sub-populations (BD Biosciences): anti-CD45-APC (clone 2D1), anti-HLA-DR-PerCP (clone L243), anti-CD14-FITC (clone M5E2), and anti-CD16-PE (clone 3G8). The samples were incubated at 4 °C for 30 min and then were treated with FACS Lysing Solution (BD Biosciences) until the erythrocytes were lysed and the cells were immediately processed in the FACSCanto flow cytometer (Becton–Dickinson Immunocytometry Systems, Palo Alto, CA) along with 10,000 beads per tube. The results were analyzed with FACSDiva Software (BD Biosciences) and the absolute numbers of lymphocytes and monocytes subsets were calculated on the basis of bead counts. Monocyte populations were classified as classical (CD14++ CD16−), intermediate (CD14++ CD16+), and non-classical (CD14+ CD16++).12 (link)