Anti human cd3 apc efluor780

Eight- to ten-week-old NOD/SCID IL2Rγ^−/− mice were intravenously injected with 1*10⁵ MOLM-13 cells. Beforehand, AML cells were cultured alone or in the presence of either purified ECAR or control vector modified T cells at an E:T ratio of 5∶1 for 4 h. Prior to the experiment, T cells were irradiated at 5 Gy to prevent xenograft reaction in recipient mice [50] . Mice were sacrificed when visible tumors developed at injection site and single-cell suspensions from bone marrow obtained from femur and tibia of the left hind leg were prepared. Erythrocytes were removed by lysis and nucleated cells were stained with anti-mouse CD45.1/PE-Cy7 (eBioscience, clone A20), anti-human CD3/APC-eFluor780 (eBioscience, clone SK7), CD19/APC (BD Bioscience, clone HIB19), CD33/PE (eBiosience, clone HIM3-4), and CD45/AlexaFluor700 (Biolegend, clone HI30) mabs. Doublet discrimination was routinely carried out and dead cells were excluded by 4, 6 diamidino-2-phenylindole (DAPI)-staining (Sigma-Aldrich, Taufkirchen, Germany). All measurements were performed on a BD LSRII FACS machine (BD Biosciences). Data analysis was realized using FlowJo-software (Tree Star Inc., Ashland, USA).

To detect human cells engrafted in HUMAMICE, multi-colors cytometric analysis was performed using BD LSR Fortessa, according to the manufacturer’s protocol but with a minor modification. HUMAMICE were sacrificed and the spleens were removed at three different times after hPBMCs were transplanted. Splenocytes were collected and lysed by ACK lysis buffer, counted and incubated with an appropriate volume of antibodies for 1 hour at 4°C and then subjected to flow cytometry analysis.
Commonly used antibodies included: Anti-Human CD45 FITC, Clone: 2D1, (ebioscience9011‐9459); isotype control, Mouse IgG1 K Isotype Control (ebioscience, FITC 11–4714) ; Anti-Mouse CD45 eFluor^® 450 (ebioscience, 48-0451-82) ; isotype control, Mouse IgG2b K Isotype Control eFluor^® 450 (ebioscience, 48-4732-82) ; Anti-Human CD3 APC-eFluor^® 780, Clone: UCHT1, (ebioscience, 47–0038) ; Mouse IgG1 K Isotype Control APC- eFluor^® 780 (ebioscience, 47–4714) ; Anti-Human CD4 PerCP-Cyanine5.5, Clone: OKT4 (OKT-4), (ebioscience, 45–0048); isotype control, Mouse IgG2b K Isotype Control PerCP-Cyanine5.5 (cat. 45–4732) ; Anti-Human CD8a PE-Cyanine7, Clone: SK1, (ebioscience, 25–0087); isotype control, Mouse IgG1 K Isotype Control PE-Cyanine7 (ebioscience, 25–4714) ; Anti-Human CD19 APC, Clone: 2H7, (ebioscience, 17–0209); isotype control, Mouse IgG1 K Isotype Control APC (ebioscience, 17–4714).