Legendplextm multi analyte flow assay kit

Anti-CaSR-Ab was purchased from Alomone Labs (Jerusalem, Israel). Anti-Caspase-12-Ab was from Abcam (Cambridge, UK). Anti-P-p65-Ab was from Cell Signaling Technology (Boston, MA, USA). Plasmid was purchased from Life Technologies (Carlsbad, CA, USA). Pacific Blue™ Annexin V Apoptosis Detection Kit with 7-AAD and LEGENDplexTM Multi-analyte Flow Assay Kit were from BioLegend (San Diego, CA, USA). RosetteSep™ Human T cell enrichment cocktail was from Stem Cell Technologies (Vancouver, WA, USA). High Pure RNA Isolation Kit and Transcriptor First Strand cDNA Synthesis Kit was from Roche (Basel, Switzerland). VPA-1002 Human T Cell Nucleofector Kit 25 test was purchased from Lonza (Basel, Switzerland).

Cytokine Bead Array (CBA) analysis was performed using the LEGENDplex^TM Multi Analyte Flow Assay Kit (BioLegend) according to the manufacturers’ instructions. For determining the cytokine concentrations in serum, 50–100 µl of whole blood was obtained 4 weeks after the immunization procedure (2 weeks before challenge with iRBCs, pre-challenge), and on day 4 after challenge with iRBCs (day 4 post-challenge) from the eye background. Plasma was obtained by centrifugation and stored at −80 °C until usage. For the analysis, 2-fold diluted sera samples and serial dilutions of the respective cytokine standards were incubated with Capture Beads and Detection Antibodies for 2 h at room temperature in the dark on a plate shaker at 600 rpm. Subsequently, Streptavidin-phycoerythrin (SA-PE) was added and samples were incubated for additional 30 min at room temperature (plate shaker at 600 rpm) followed by a washing step with Wash Buffer. Finally, the bead pellet was re-suspended in Wash Buffer for measurement. 300 events/cytokine were acquired on a FACS Calibur. Cytokine concentrations were analyzed using the LEGENDplex^TM Data Analysis Software Version 7.0 (Biolegend).

Cytokine quantification was realized using the LEGENDplex^TM multi-analyte flow assay kit (Biolegend, San Diego, CA, USA). Sera from humanized mice were incubated for 2 h at room temperature with customized pre-mixed beads and detection antibodies specific for a panel of 13 human cytokines (IL-23, IL-12, IFNγ, TNFα, MCP-1, IL1β, IP-10, IL6, IFNβ, GM-CSF, IFNα2, IL18, IL33). Samples were then incubated for 30 min with SA-PE (Biolegend), wash and resulting fluorescent signals were analyzed on a flow cytometer (LSRII, BD Biosciences) according to manufacturer’s instructions. Analyte concentration was determined using LEGENDplex^TM software (Biolegend, San Diego, CA, USA).

Mouse serum levels of cytokines, including IL-6, CCL5, CXCL10, TNF-α, and IFN-α, were detected using LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, cat.no. 740625) according to manufacturer’s instructions. The samples were acquired on a BD LSRFortessa^TM Flow Cytometer (BD Biosciences) applying a 488 nm laser with 536/40 (BP) filter for the PE fluorochrome and a 638 nm laser with 675/20 (BP) for the APC fluorochrome. The results were analyzed using LEGENDplex™ Data Analysis Software V.8.0 (Vigene Tech Inc., USA). The concentration of each growth factor/cytokine was determined by means of a standard curve generated during the performance of the assay.
The levels of white blood cells, lymphocytes cells, and monocytes cells in mouse blood were measured according to the manufacturer’s protocol by Shanghai Model Organisms Inc.

Cytokines IFN-γ, TNF-α, IL-2, IL-5, and IL-6 in tumor tissues extracted from either the Control group or the P5091 group were detected by flow cytometry according to LEGENDplex^TM Multi-Analyte Flow Assay Kit (740750, Biolegend).

The secretion of anti-tumor cytokines from SN-derived T cells with SIGLEC15 knockdown was analyzed using LEGENDplex^TM Multi-Analyte Flow Assay Kit (BioLegend, San Diego, CA, United States) as described previously (Wallrapp et al., 2017 (link)). Single-cell suspension prepared from the SN of five CRC patients were cultured in serum-free medium and transfected with SIGLEC15-specific siRNA or control siRNA for 72 h. The transfected cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin overnight, and the production of interleukin-2 (IL-2), Interferon-γ (IFN-γ), and tumor necrosis factor alpha (TNF-α) in the supernatant was determined by flow cytometry.