Cd226 fitc

Lymphocytes isolation and identification was described as our previous study [34 (link)]. Briefly, Lymphocytes was isolated from human blood using the human lymphocyte separation medium, then transferred to a 25 cm² plate (Costar), precoated with RetroNectin (2 mg/L; TaKaRa, Japan) and anti-CD3 antibody (50 ng/ml; TaKaRa, Japan) at 1 × 10⁶ cells per well, and incubated for 48 hours for optimal activation before transduction. The stimulated CTL lines were resuspended at 1 × 10⁶ cells/mL in complete medium supplemented with IL-2 (300 U/ml), IFN-c (400 U/ml), phytohaemagg lutinin (10 U/ml), rhIL-4 (100 U/ml), GM-CSF (10 U/ml) and rhIL-2 (100 IU/mL), and then incubated for 36 hours at 37°C and 5% CO₂. CD3-FITC, CD4-PE, CD8-PE, CD56-PE, CD226-FITC, CD11-PE and CD305-FITC monoclonal antibodies (MAbs) with the appropriate fluorescein isothiocyanate (FITC)- or R-phycoerythrin (PE)-conjugated were purchased from BD Bioscience (Mountain View, CA). Cells were washed and stained with the appropriate antibodies for 20 minutes at 4°C in the dark in phosphate-buffered saline (PBS) supplemented with 0.1% bovine serum albumin (BSA). Control cells were unstained. After incubation, the cells were washed twice and resuspended in PBS.

Single-cell suspensions were stained according to standard protocols and subsequently stained for 30 min at 4°C with the following antibodies (BD Biosciences): TIGIT-FITC, CD226-FITC, PD1-FITC, CD155-PE, CD33-APC, CD34-FITC, CD3-PerCP, CD4-APC, CD8-PE, CD16-APC, CD56-BV421, and CD56-PB450. CD56⁺ NK cells were divided into CD56⁺CD16^– NK (CD56^bright NK) cells and CD56⁺CD16⁺ NK (CD56^dim NK) cells using an anti-human CD16 antibody (24 (link)). After washing the suspension twice, the cells were analyzed by FCM. The fluorescence compensation between channels was adjusted to circle the target cell group, and the FCM data were subsequently analyzed using Cell Quest^TM Pro 4.0.2 software (BD Biosciences).

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).