Fitc nkp46

For analysis of pluripotency markers, iPSCs were dissociated into single cells using TrypLE^TM Select (Gibco), blocked with 10% human AB serum, stained with Alexa Fluor 488-SSEA-3 and Alexa Fluor 647-TRA-1-60 (BioLegend, San Diego, CA, USA), and analyzed by BD FACS Canto flow cytometer (BD Biosciences) with FlowJo software (V10.4.1). For phenotypic analysis of HPC induction and NK commitment, the cells were resuspended in FACS buffer (PBS with 2% bovine serum albumin), incubated with antibody cocktail for 30 min at 4 °C in the dark, and analyzed by BD FACS Canto flow cytometer. The antibodies used in this study included FITC-CD16, FITC-Nkp46, PE-CD56, PE-Nkp44, PerCP-CD45, PerCP/Cy5.5-CD94, APC-CD34, PE/Cy7-CD43, PE/Cy7-KIR, Alexa Fluor 700-CD161, and BV605-Nkp30 (BioLegend).

After cocultivating NK cells and PC cells with different treatments for three days, NK cells were collected and washed twice with PBS, and then, anti-human CD3-PerCP/Cyanine5.5, CD16-PE, CD56-PE, NKG2D-APC, NKp46-PE/Cyanine7, and DNAM-1-FITC antibodies were added (BioLegend, San Diego, CA, USA) according to the instructions. The cells were incubated for 20 min in the dark and washed twice with PBS. For the detection of NK cells in mouse peripheral blood, 100 μl mouse peripheral blood was collected, and anti-mouse CD3-PerCP/Cyanine5.5, NK1.1-PE-Cyanine7, NKG2D-APC, NKp46-FITC, and DNAM-1-PE antibodies were added (BioLegend, San Diego, CA, USA) and incubated at room temperature for 20 minutes in the dark. Then, 2 ml RBC lysis solution was added to each tube, mixed well, and incubated for 15 min. Then, the samples were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. All samples were then analyzed by multicolor flow cytometry (BD, USA).