Cd3 pe cy7 clone

The following directly labeled antibodies were used for FACS analysis CD45 APC-Cy7 clone: H130 (1 mg/ml, 1:200), CD3 PE-Cy7 clone: UCHT1 (1 mg/ml, 1:100), HLA-DR PE clone: LN3 (1 mg/ml, 1:400), CD68 PerCp-Cy5.5 clone: RPA T8 (1 mg/ml, 1:50), CD303 APC clone: 201A (1 mg/ml, 1:100)–all from Biolegend; CD19 PerCp-Cy5.5 clone: HIB19 (1 mg/ml, 1:50), CD11c APC clone: Bu15 (1 mg/ml, 1:100), CD14 PE-cy7 clone: 61D3 (1 mg/ml, 1:200), CD16 PE clone: B73.1 (1 mg/ml, 1:200), CD1c FITC clone: L161 (1 mg/ml, 1:50), DC-SIGN PE-Cy7 clone: eB-h209 (1 mg/ml, 1:100)–all from ebioscience; CD11b Pacific blue clone: CBMR1/5 (1 mg/ml, 1:50) and CD11b Alexa 700 clone: CBMR1/5 (1 mg/ml, 1:300)–from BD bioscience; HLA-DR FITC clone: AC122 (1 mg/ml, 1:400) and CD141 PE clone: AD5-14H12 (1 mg/ml, 1:50)–from Miltenyi Biotec; and 25F9 FITC clone: HM2158F (1 mg/ml, 1:50) from Hycult.

Bone marrow and liver MNCs were harvested from the mice. Single-cell suspensions were blocked with an Fc receptor CD16/CD32 (0.5 μL/10⁶ cells) at room temperature. After 10 min, the cells were stained with a cocktail of antibodies. After 30 min of staining at room temperature, the cells were washed with a 1 × phosphate buffer solution.
Monoclonal antibodies for murine lineage antibodies cocktail comprised of Peridinin chlorophyll protein complex (PerCP)-cy5.5 (clone, 51-9006977), CD127-Phycoerythrin (PE)-Cy7 (clone, A7R34), marker of proliferation Ki-67 (Ki67)-PE (clone, SolA), CD4-Percpcy5.5 (clone, RM4-5), and CD8aα-Percpcy5.5 (clone, 53-6.7) were from BD Biosciences (San Diego, CA, USA). Anti-mouse CD45-Percpcy5.5 (clone, 30-F11), CD48-Allophycocyanin (APC)-Cy7 (clone, HM48-1), CD135-PE (clone, A2F10), CD34-Fluorescein isothiocyanate (FITC) (clone, RAM34), CD44-FITC (clone, IM7), CD3-PE-cy7 (clone, 145-2C11), T cell receptor gamma delta (TCRγ/δ)-APC (clone, GL3), Notch1-BV421 (clone, HMN1-12), Notch2-APC (clone, HMN2-35), and stem cell antigen 1 (Sca-1)-APC (clone, D7) were purchased from BioLegend (San Diego, CA, USA). CD117 (c-Kit)-PE-eFluor610 (clone, 2B8), CD25-PE (clone, PC61.5), and TCRβ-PE-eFluor610 (clone, H57-591) were purchased from eBioscience (San Diego, CA, USA).

To measure the gene expression on both mRNA electroporated primary NK cells and lentiviral transduced primary NK cells, the cells were first collected and then washed once in 1 X PBS + 1% BSA. The cells were then incubated with 6 ng/mL CD73, Avi-His-Tag biotin labeled recombinant protein (BPS Bioscience) for 30 min at 37 °C. After the first incubation, cells were washed and stained with surface markers, CD56 (PE, clone: CMSSB), CD3 (PE-CY7, clone:UCHT1), and Strep-BV421 (Biolegend) and incubated for 30 min at 4 °C. The cells were then washed and the Sytox green dead cell stain (Thermo Fisher) was added for cell viability. The gene expression was then detected via flow cytometry. Cells were also sorted by staining the cells with an anti-CD73 avi-His-Tag biotin labeled antibody and Strep-BV421 antibody at the Purdue Flow Cytometry Core to measure gene expression both before and after sorting.