Cd96 pe

NK cells were incubated with optimal concentrations of the following anti-human Abs: CD56-PE, CD3-PerCP, NKG2D-APC, CD226-FITC, CD96-PE or the corresponding isotype control Abs (all from BD Pharmingen) in 100 μl of 1% FCS/PBS at 4°C for 20 min.
Tumour cells, with and without platelets, were incubated with anti-human MICA, MICB unconjugated antibodies and alexa-fluor-488 secondary antibody. Staining of tumour cells using primary and secondary antibodies was performed for 1 hour at 4°C. Tumour cells were also stained with CD112-APC and CD155-FITC antibodies and isotype controls in 100 μl of 1% FCS/PBS at 4°C for 20 min.
Cells were washed twice with PBS and acquired on a Cyan flow cytometer (Beckman Coulter, Brea, CA, USA). Events were stored and analysed on the FlowJo software (TreeStar). NK cells were gated from PBMC populations as distinct CD3-CD56+ cells (S1 Fig).

First, 100 μl of bone marrow (BM) samples were collected from MDS patients or HCs. Then, the bone marrow aspirations were stained with the following antibodies (BD Biosciences) at 4 °C for 15 min in the dark. Then 2 ml of lysing solution (BD Biosciences) was added to each tube to lyse erythrocytes for 10 min in the dark at room temperature, centrifuged at 1500 rpm for 5 min, and washed twice with phosphate-buffered saline (PBS). Finally, we analyzed the cells by FCM after washing the cells twice with phosphate-buffered saline (PBS). CytExpert Software (Beckman CytoFLEX) was used to analyze the FCM data.
The following antibodies (BD Biosciences) were used in this study: CD3-PerCP, CD56-APC-Cy7, TIGIT-FITC, CD226-APC, CD96-PE, NKG2D-PE-Cy7, CD107a-Bv421, IFN-γ-PE-Cy7, Perforin-Bv421, CD226-FITC, TIM-3-APC-Cy7, CD96-PE-Cy7, VISTA-PE-Cy7, PD-1- Bv421, LAG-3-Bv421, CD4-APC, CD8-PE, and CD56-PE. NK cells were labeled with CD3⁻CD56⁺, CD4⁺T cells with CD3⁺CD4⁺, and CD8⁺T cells with CD3⁺CD8⁺.