Anti cd3 pe sk7

Human AML cells were treated with indicated drug for 2 h, followed by extensive washing. Three days later, the cells were stained with anti-CD45-V500 (2D1, BD Bioscience, 1:20), anti-CD34-BV421 (581, BD Bioscience, 1:20), anti-CD38-APC (HB7, BD Bioscience, 1:50), anti-CD33-PE-Cy7 (p67.6, BD Bioscience, 1:20), anti-CD3-PE (SK7, BD Bioscience, 1:50), anti-CD19-APC-H7 (SJ25C1, BD Bioscience, 1:10), 7AAD (BD Bioscience, 1:10), and anti-CD11b-FITC (Bear1, BD Bioscience, 1:10) or anti-CD7-FITC (M-T701, BD Bioscience, 1:20) for 30 min. Then 15 µL of well-suspended flow count fluorospheres (Beckman Coulter) were added right before analysis by flow cytometry with BD Fortessa.

Samples from the confirmation cohort were fractionated and lysed fresh and immediately after PBMC isolation fractionated into monocytes and the remaining PBMC fraction with EasySep Human CD14 positive selection kit II (STEMCELL Technologies, Vancouver, BC, Canada) according to the manufacturer's instructions. The purity of the monocyte and remaining lymphocyte fractions was confirmed by flow cytometry (Table S2). Both cell fractions and unfractionated PBMC were immunostained with anti‐CD3 PE (SK7, BD Biosciences, San Jose, CA, USA) and anti‐CD19 APC (SJ25C1, BD Biosciences) for 30 min at +4°C. The PBMC and remaining fraction were furthermore stained with anti‐CD14 FITC (M5E2, BD Biosciences) for 30 min at +4°C to assess the initial and remaining amounts of monocytes in the sample. After immunostaining the cells were washed twice with phosphate buffered saline (PBS) for 5 min at 2500 rpm with Sorvall MC 12 V (Thermo Fischer Scientific, USA). The cells were fixed with 0.1% formaldehyde in PBS. The samples were analyzed using an Accuri C6 flow cytometer (BD Biosciences). Fractionated cells were lysed in Buffer RLT Plus (Qiagen) and stored at −80°C prior to RNA extraction.