Alexa 647 labeled streptavidin

To detect cell surface expression of CD3E and CD3D proteins on TCR-KO Jurkat cells, TCRβ-T2A-TCRα-pEF1α-IRES-hKO1 vectors (2 μg) were transfected into TCR-KO Jurkat cells (1 × 10⁶ cells/sample, 100 μL/well) by using the 4D-Nucleofector electroporator (Lonza, program: CL-120) and SE Cell Line 4D-Nucleofector X Kit L (Lonza) according to the manufacturer’s instruction.
Two days later, transfected cells were harvested and stained with anti-CD3E-PE-Cy7 (clone UCHT1, BioLegend) and anti-CD3D-APC (clone 7D6, Invitrogen). Biotinylated anti-CD3E antibody OKT3 (BioLegend) was diluted to 200 μg/mL and serial dilution was performed (1:5). Diluted antibody solution (100 μL/well) was transferred to a 96-well plate and mixed with equal amount of TCR-transfected TCR-KO Jurkat cells (1 × 10⁵ cells/sample, 100 μL/well). After incubation (4°C, 30 min), cells were washed twice and stained with Alexa 647-labeled streptavidin (BioLegend, diluted in 1:400, 50 μL/sample). Samples were prepared as described in the “flow cytometry” section and analyzed using a BD LSRII (BD Bioscience).

Lung sections from M.tb infected mice were subjected to deparaffinization and antigen retrieval prior to blocking for 1 h with 10% goat serum and 1% FBS in a humidified chamber (Wu et al., 2019 (link)). The sections were incubated at 4°C with primary antibodies against CD4 (rabbit mAb D7D2Z, Cell Signaling), LAG3 (rat mAb C9BZW, BioLegend), CD49b (biotin labeled rat mAb DX5, BioLegend), and IL-10 (rat Mab MT60, MABTECH). After washing PBS were incubated for 1 h with secondary antibodies Alexa 488 labeled donkey F(ab´)₂ anti-rabbit IgG and Alexa 647 labeled donkey F(ab´)₂ anti-rat IgG (Abcam) and Alexa-647 labeled streptavidin (BioLegend). Nuclei were stained with DAPI. All fluorescence images were captured using an Olympus FV 1000 Spectral Confocal system. Mean fluorescence intensity of cells was determined using Image J (version 2.0.0).