Ifn γ elisa kit

Splenocytes were prepared and counted from each mouse in different groups as described previously (32 (link)). Subcomponent-specific cytokines Th1 (TNF-α and IFN-γ), Th2 (IL-4), and Th17 (IL-17A) were detected using cytometric bead array (CBA). 5 × 10⁶ cells were plated into each well of a 24-well plate and incubated with RPMI1640 medium (negative control), or RPMI1640 medium with specific proteins including Rv0577, Rv2875, Rv3044, or Rv2073c (each 10 µg/mL), respectively. The supernatant was collected 24 h later and Cytometric Bead Array kit (BD Biosciences, NJ, USA) was performed according to the manufacturer’s instructions.
Alternatively, 5 × 10⁶ cells were incubated with CMFO protein (10 µg/mL) at 37°C and 5% CO₂. RPMI1640 medium was used as a negative control and PPD (10 mg/mL, Statens Serum Institut, Denmark) was set as a positive control. After an incubation of 72 h, the supernatant was collected and a commercial mouse IFN-γ ELISA kit (Multi Sciences, Hangzhou, China) were used to detect the concentration of IFN-γ (20 (link)–22 (link)). The results are expressed as mean ± SD (pg/mL) for each group (n = 6).

The quantified levels of IFN-γ, TNF-α, Granzyme B secreted by co-cultured activated CD8⁺ T cells were detected using ELISA kits, including Human TNF-α ELISA Kit, IFN-γ ELISA Kit (MultiSciences, China) and Human Granzyme B ELISA Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s protocol. The supernatants were collected and detected by 450 nm Optical densities using microplate reader (Bio-Tek). For the surface PD-L1, ~ 80% confluency BC cells were stained by FAM-labeled PD-L1 antibody (BioLegend, USA). After washing by PBS three times, FAM-positive cells were detected by flow cytometry. The release of lactate dehydrogenase (LDH) in the supernatants was detected on cell lysis using the Cytotoxicity Detection Kit PLUS (Sigma-Aldrich) according to the manufacturer’s protocol.

In a U-bottom 96-well plate, the CD4⁺ T cells expressing the sdCAR were incubated with cognate or non-cognate antigen-expressing target cells at an effector:target cell ratio of 1:2 in varying types of switch molecules including PBS, HM-3, and FHBM, respectively. Simultaneously, a positive control was designed, including a second generation of CAR specific for MSLN (MζBB CAR), to assess the sdCAR-T cell cytotoxicity. After incubation for 24 h, the supernatant was harvested, and the levels of interleukin-2 (IL-2) or interferon γ (IFNγ) were measured using the human IL-2 or IFNγ ELISA kit (MultiSciences), respectively. At the same time, the cells were resuspended in PBS and stained with an APC-conjugated anti-human CD69 antibody (BioLegend). Stained cells were washed three times with PBS and finally resuspended in 200 μL PBS. CD69 expression was detected with a flow cytometry.