Anti cd3 clone ucht1

After thawing, PBMC were washed and immediately stained with the following panel: anti-CD3 (clone UCHT1; #IM2467, Beckman Coulter, Brea, CA, USA), anti-CD4 (clone SFCI12T4D11; #737660, Beckman Coulter), anti-CD8 (clone RPA-T8; #558207, BD Biosciences, Franklin Lakes, NJ, USA), anti-CD14 (cloneMφP9; #641394, BD Biosciences), anti-CD16 (clone3G8; #555406, BD Biosciences), anti-CD56 (clone N901; #A07788, Beckman Coulter), anti-CD11c (clone B-ly6; #561352, BD Biosciences), anti-CD19 (clone SJ25C1; #563036, BD Biosciences), anti-CD123 (clone 6H6; #45-1239-42, eBioscience, Affymetrix, Santa Clara, CA, USA), anti-HLA-DR (clone Immu-357; #IM3636, Beckman Coulter), and Zombie UV (#77474, Biolegend, San Diego, CA, USA). The samples were acquired on an BD Fortessa instrument equipped with the FACS Diva software. The analysis was performed with the FlowJo (FLOWJO, LLC, Ashland, OR, USA).

After enrichment, cells were washed and incubated at a dilution of 1/50 with mouse serum (Sigma-Aldrich) for 10 min at 4 °C. Cells were stained with anti-CD3 (clone UCHT1, FITC, Beckman Coulter), anti-CD14 (clone RMO52, FITC, Beckman Coulter), anti-CD19 (clone J3-119, FITC, Beckman Coulter) anti-CD45 (clone HI30, APC-Cy7, Biolegend) and anti-CD56 (clone N901(NKH-1), PE, Beckman Coulter) antibodies for 30 min at 4 °C. We added an anti-CD34 (clone 581, FITC, BD Biosciences) antibody to the lineage to sort AML samples. Cells were then washed twice with DPBS and incubated in DPBS with a dead cell marker for 10 min at 4 °C. Cells were washed again and sorted in an Influx or Melody cell sorter from BD (Becton Dickinson, San Diego, USA).

10⁵ peripheral blood monocyte cells (PBMC) from healthy consenting donors were stimulated with phytohemagglutinin (PHA, 1μg/ml, Invitrogen) and exposed to supernatant of 24-hours cultured cells initially seeded at 4.10⁴cells/ml. After 4 days-exposure, PBMC were overnight exposed to 1μCi ³H-thymidine (Perkin-Elmer, Groningen, The Netherlands) prior to measure incorporated radioactivity (TopCount, Perkin-Elmer, Meriden, CT, USA).
In other experiments, 10⁵ PBMC were stimulated with anti-CD3 (clone UCHT1, 2μg/ml, Beckman Coulter, Villepinte, France) and anti-CD28 (clone CD28.2, 2μg/ml, Beckman Coulter) antibodies and labeled with CFSE (1μM, Invitrogen). Stimulated PBMC were then exposed to supernatants collected from fibroblasts. After 4 days, the decrease in CFSE fluorescence assessed by flow cytometry was used to determine PBMC proliferation.