CAR-T cells were sorted by the FACS Aria cell sorting system (FACSCalibur, BD) using GFP. To assess CAR-T cell cytotoxicity, Nalm6-GL and K562-GL cell lines expressing luciferase were plated at 2 × 104 cells/well in black 96-well plates (Corning Costar, Corning, NY, USA) and cultured with T cells/well in an effector to a target ratio of 1:1, 1:2, 1:4, 1:8, and 1:16. Before coculture, T cells were washed three times with phosphate-buffered saline. After 18 h of being cocultured with Nalm6-GL, the medium was aspirated for IL-2, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) secretion analysis using the BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (BD Biosciences). The viability of the cancer cells was determined by measuring luminescence with a GloMax Discover Plate Reader (Promega Madison, WI, USA) after adding luciferin (150 μg/ml) to every well.
Cytometric bead array cba human th1 th2 cytokine kit 2
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The BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II is a flow cytometry-based assay designed to simultaneously measure multiple human cytokines. The kit utilizes a panel of beads coated with capture antibodies specific for different cytokines, allowing for the quantification of IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ in a single sample.
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9 protocols using cytometric bead array cba human th1 th2 cytokine kit 2
1
Quantifying CAR-T Cytotoxicity and Cytokine Profiles
2
Quantifying Human Cytokine Levels
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The amounts of human cytokines including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-2, IL-4, and IL-10 in plasma were quantified by using BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (BD Biosciences) according to the manufacture’s protocol23 (link).
3
Immunological Assessment of IRE and NK Therapy
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One to four days prior to IRE treatment, and 1 month following the final NK cell transfusion, peripheral blood (2 ml) was collected and assessed using flow cytometry (FACSCanto™ II; BD, Grand Island, NY, USA). The tested indices included lymphocyte number and function in the patients’ peripheral blood. A BD Multitest 6-color TBNK reagent (No. 644611) was used to detect the number of CD3 + CD4 + cells, CD3 + CD8 + cells, total CD3 + cells, CD3 − CD19 + cells, and CD3 − CD16 + CD56 + cells. The BD cytometric bead array (CBA) human Th1/Th2 cytokine kit II (No. 551809) was used to detect the expression levels of interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor (TNF), and interferon-γ (IFN-γ). The tests were performed according to the protocols in the instruction manuals. Results above or within the reference range were defined as normal; results below the reference range were defined as immune dysfunction. For the IRE group, peripheral blood was drawn 1–4 days prior to IRE and 1 month following IRE; blood was drawn from the IRE–NK group 1–4 days prior to IRE and 1 month following NK cell therapy.
4
Immune Dysfunction Evaluation via Multiparameter Flow Cytometry
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2 ml peripheral blood from patients was drawn 1 day before cryoablation and 3 days after cryoablation or one course NK cells therapy (Figure 1B ). BD Multitest 6-color TBNK Reagent (BD Biosciences, USA) was used to detect the number of CD3+CD4+ cells (95% range: 441–2156 cells/μl), CD3+CD8+ cells (95% range: 125–1312 cells/μl), total CD3+ cells (95% range: 603–2990 cells/μl), CD3-CD19+ cells (95% range: 107–698 cells/μl) and CD3-CD16+CD56+ cells (95% range: 95–640 cells/μl). BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (BD Biosciences, USA) was used to detect the expression levels of IL-2 (95% range: 8–12.5 pg/ml), IL-4 (95% range: 3.5–6 pg/ml), IL-6 (95% range: 2.7–8.5 pg/ml), IL-10 (95% range: 1.8–4 pg/ml), tumor necrosis factor (TNF; 95% range: 1.7–2.5 pg/ml) and IFN-γ (95% range: 1.5–4 pg/ml). The number and function of lymphocytes in the peripheral blood of patients were tested according to the protocols given in the instruction manuals. Results above or within the reference range were considered to indicate normal immune function. When one or more values were below the reference range, it was considered to indicate immune dysfunction.
5
Multiwalled Carbon Nanotube Induced Cytokine Levels
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A commercial BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (lot number: 551809, BD Biosciences Pharmingen, San Diego, USA) was used to determine the levels (pg/ml) of Th1 and Th2 cytokines, including Interferon-gamma (IFN-γ), tumor necrosis factor (TNF), interleukin (IL)-10, IL-6, IL-4 and IL-2 in A549 supernatant samples following the manufacturer's instructions, after treatment of MWCNT at 0.3, 3, 30 µg/ml for 2, 12 and 24 h. A standard calibration curve was established for each kit. The maximum and minimum limits of detection for all six cytokines were 1.0 and 5000 pg/ml, respectively. Fluorescence was analyzed using a flow cytometer (FACS Calibur, Becton-Dickinson Biosciences, Heidelberg, Germany) and cytokine level was determined using a BD CBA Software. Statistical analysis was performed using a Kruskal-Wallis test (nonparametric analogue of one-way ANOVA) for the data that did not meet the assumptions of parametric tests. The differences were considered significant at p<0.05.
6
Cytokine Profiling of Cell Supernatants
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Supernatants were evaluated for interleukin (IL)-2, IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNFalpha) and interferon gamma (IFNgamma) content using the BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (BD Biosciences) performed according to the manufacturer’s instructions, measured by FACS and analyzed by FCAP Array v3.0 software (all BD Biosciences).
7
Serum Cytokine Profiling in Mice
8
Cytokine Secretion Assay of OKT-CIK and R-CIK Cells
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After 15 days in culture, OKT-CIK and R-CIK cells (1 × 105) were plated at 1 × 105 cells per well in a 96-well flat-bottom plate, with 1 × 105 K562 tumor cells and 200 μL GT-T551 medium without IL-2. After 24-hour coculture, supernatants were harvested and cytokine secretion was quantified by BD Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II (BD Biosciences, San Jose, CA, USA) according to the protocol of the kit.
9
Quantitative Cytokine Profiling of Gene-Edited PBMCs
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One day after electroporation (10×10 6 cells in 2 mL, 1 mL per well), gene-edited and control PBMCs were incubated with PMA/ionomycin (eBioscience stimulation cocktail) for 4 hours at 37C°. Subsequently, supernatant was collected, and stored at -80°C.
The human Th1/Th2 Cytokine Assay (BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II) was used to quantitatively measure cytokine production according to the manufacturer's protocol. In brief, supernatant was thawed and 50 μL was used for the cytokine bead array. Samples and standards were incubated with the capture beads for 3 hours at room temperature, while protected from light (Supplementary data, supplementary table 2). After incubation, assay tubes were washed, centrifuged and resuspended before flow cytometer analysis.
The human Th1/Th2 Cytokine Assay (BD™ Cytometric Bead Array (CBA) Human Th1/Th2 Cytokine Kit II) was used to quantitatively measure cytokine production according to the manufacturer's protocol. In brief, supernatant was thawed and 50 μL was used for the cytokine bead array. Samples and standards were incubated with the capture beads for 3 hours at room temperature, while protected from light (Supplementary data, supplementary table 2). After incubation, assay tubes were washed, centrifuged and resuspended before flow cytometer analysis.
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