Apc conjugated cd69 antibody

Jurkat cells were washed three times with PBS and incubated for 30 min at 4 °C with saturating concentrations of allophycocyanin (APC)-conjugated CD69 antibody (BioLegend, CA, USA). After two washes, the cells were resuspended in PBS. Negative control cells were incubated with APC-conjugated human IgG. The immunofluorescence intensity of cells was determined by a Coulter-EPICS XL flow cytometer (Beckman, FL, USA) and analyzed using FlowJo software (Treestar, Inc., CA, USA).

For the flow cytometry assays, all cells were harvested and washed three times with FACS wash buffer (1 × PBS containing 0.5% BSA and 0.03% sodium azide) prior to flow cytometry. Surface staining of cells was performed with monoclonal antibodies directed against CD4, CD8, and CEA, which were purchased from Becton Dickinson (BD), while the monoclonal antibody against MSLN was purchased from R&D Systems. Transgenic populations with GFP expression for T cells or with RFP expression for tumor cells were analyzed on the fluorescein isothiocyanate (FITC) channel or the PerCP channel, respectively. For T cell activation, after overnight incubation, the T cell surface markers, CD25 and CD69, were detected using the APC-conjugated CD25 antibody (Biolegend) and APC-conjugated CD69 antibody (Biolegend), respectively. After a 30-min incubation period at 4 °C, protected from light, all cells were washed and analyzed by flow cytometry.
For western blot analysis, the proteins were extracted from modified cells and stored at − 80 °C. Western blots were performed using GFP primary antibody (abcam) and RFP primary antibodies (abcam).

For flow cytometry, cells were collected by centrifugation and washed three times with FACS wash buffer (1x PBS with 0.5% BSA, and 0.03% sodium azide). Cell surface was performed using an anti-AXL antibody (Becton Dickinson (BD), San Jose, CA, USA). Regarding the evaluation of T cell activation, T cell surface marker CD69 was detected using an APC-conjugated CD69 antibody (Biolegend, San Diego, CA, USA) after overnight incubation of effective target cells. The phenotypes of T cells were determined using a FITC-conjugated CD4 antibody (BD) and a PE-conjugated CD8 antibody (BD). After incubation for 30 minutes at 4°C in the dark, cells were washed and analyzed by flow cytometry.