Cell were collected and split in half. One half was stained with Alexa594-conjugated mouse anti-GFP antibody (Thermo) and Alexa647-conjugated anti-NGFR (BD Biosciences) while the other half were stained with Alexa594-conjugated mouse anti-GFP antibody and Alexa647-conjugated anti-CD19 antibody (Thermo). Cells were stained for 30min at 4°C and subsequently washed with ice old DPBS. Cells were then fixed with Cytofix (BD Biosciences) for 10min at room temperature, washed with DPBS and loaded onto a glass slide using Cytospin Cytocentrifuge (Thermo). Prolong Diamond Antifade Mountant with DAPI (Thermo) was applied before sealing the slide with a category 1.5 cover slip (Thermo). The slides were imaged using Zeiss LSM710 and image data were analyzed with FIJI (Image J).
To quantify binding of p40-GFP fusion protein on cell surface, the cell membrane was first defined based on Alexa647 signal (anti-NGFR or anti-CD19 that marks p40-Td or ΔCD19-Td cells, respectively). In parallel, an irrelevant area (either area with no cell or intracellular space) were selected as background area. The mean fluorescence intensity (MFI) of Alexa594 signal (anti-GFP) was measured on membrane area and background area and the surface binding of p40-GFP was calculated as MFI(membrane)-MFI(background).
To quantify binding of p40-GFP fusion protein on cell surface, the cell membrane was first defined based on Alexa647 signal (anti-NGFR or anti-CD19 that marks p40-Td or ΔCD19-Td cells, respectively). In parallel, an irrelevant area (either area with no cell or intracellular space) were selected as background area. The mean fluorescence intensity (MFI) of Alexa594 signal (anti-GFP) was measured on membrane area and background area and the surface binding of p40-GFP was calculated as MFI(membrane)-MFI(background).