Canto 2 flow cytometer

The ROS inhibitory activity of 5-TDMF was measured as described by Bass [35 ]. The BNLCL2 Cells (1 × 10⁵ cells/mL) were co-cultured with or without 5-TDMF (32 and 64 μM) for 24 h. NAC was taken as positive control. After incubation, 0.1 mM H₂O₂ was added and the mixture was incubated at 37 °C for 1 h then culture medium was replaced with fresh medium containing 10 μM DCFH-DA. After 30 min of incubation at 37 °C, fluorescence intensities of DCF were measured on a Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). To measure the 5-TDMF-induced GSH levels, the o-phthalaldehyde (OPA) conversion method by Senft was used [36 (link)]. 50 μg/mL of OPA in 0.1 M Na₂HPO₄-5 mM EDTA buffer was added to the 5-TDMF-treated cells and the reaction was kept in the dark at 25 °C for 1 h. After two wash steps with phosphate buffer (pH 7.0), the intensity of GSH fluorescence was analyzed on a Canto II flow cytometer (BD Biosciences).

For cell growth analyses cells were seeded in 96-well plates and allowed to attach overnight. After treatment, cell density was analyzed by CV staining after the indicated intervals as previously described^{42 (link),47 (link)}. Cell viability was analyzed by lactate dehydrogenase release assays with the Cytotoxicity Detection Kit (LDH) (Roche, Mannheim, Germany) or by propidium iodide (PI) uptake assays via flow cytometry as previously described^{30 (link),33 (link)}. Data were acquired with a BD Canto II flow cytometer and analyzed with BD FACS Diva software (version 6.1.3).

Flow cytometry was performed on a BD Canto II flow cytometer and analyzed using the FlowJo software (BD Biosciences). A list of antibodies used here was summarized in Table S2. For intracellular staining of IFN-γ, fresh isolated cells are treated with phorbol 12-myristate 13-acetate/ionomycin cocktail according to the manufacturer’s specification (BioLegend). The cells were then washed, stained with antibodies against CD3, CD4, and CD8α, fixed with fixation buffer and subsequently stained intracellularly with antibodies against IFN-γ in Intracellular Staining Permeabilization Wash Buffer (BioLegend). Doublets and debris of dead cells were excluded before various gating strategies were applied. Gates and quadrants were set based on isotype control staining, and the mean fluorescence intensity (MFI) values are calculated by minus the MFI of isotype control antibodies.

Yeast cells displaying HA from A(H1N1)pdm09 were incubated at 60°C for 30 minutes. The sample was then chilled on ice for 30 minutes and separately labelled with 100nM of R1a-F5, R1a-G6, R2b-E8, R2b-D9, R1a-A5, R1a-B6 and R2a-G8, or 1μg/ml of mouse anti-SV5 antibody. After washing, cells were stained with secondary reagents as previously described and mean fluorescence intensity (MFI) determined. The head-binding single domain antibody R1a-F5 was sub-cloned into the yeast display vector pNIBS-5 as a Sfi1/Not1 fragment and transformed into EBY100. The antigen standard A/California/07/2009 (H1N1)pdm09 at 100nM concentration in either 50mM Tris-HCl pH 8.0 or 100mM sodium acetate buffer pH4.8 was pre-incubated for 1 hour at room temperature followed by incubation with yeast cells displaying R1a-F5. After a further 30 minutes, cells were incubated at neutral pH containing sdAbs at 1μg/ml in PBS. sdAb binding was then assessed by labelling with anti c-Myc as described previously and analysis on a BD Canto II flow-cytometer.

After transfection with miR-34a and NC-RNA mimics for 48 h, MDR-MCF-7 breast cancer cells were gently scraped, washed twice with chilled PBS (4°C), and suspended at a concentration of l × 10⁶ cells/mL for flow cytometry detection. For cell cycle analysis, target cells were fixed in 75% ethanol and stained with propidium iodide (Sigma Aldrich) supplemented with RNase A (Roche). The Annexin V-APC/7-AAD apoptosis kit (KeyGEN Biotech, Nanjing, China) was used for apoptosis assays. Each set of two wells was analyzed 3 times with a BD Canto II flow cytometer (BD Biosciences, San Jose, CA, USA). Data were analyzed using CellQuest software.

Freshly obtained intestinal biopsy samples were immediately cultured in complete medium (1 biopsy per 1 mL of complete medium per well) for 24 hours, after which culture media were filtered (0.45 μm) and cell-free supernatants preserved (−80°C) until they were analyzed using a FlowCytomix Multiple Analyte Detection (eBioscience, Wien, Austria) on a BD CantoII flow cytometer (BD Biosciences), following the manufacturer’s instructions, for the concentration of interferon (IFN) γ (detection limit [DL]: 0.25 pg/mL), IFNα (DL: 4.04 pg/mL), interleukin-1β (IL-1β) (DL: 1.80 pg/mL), IL-2 (DL: 0.35 pg/mL), IL-4 (DL: 1.22 pg/mL), IL-5 (DL: 0.76 pg/mL), IL-6 (DL: 4.10 pg/mL), IL-7 (DL: 3.18 pg/mL), IL-9 (DL: 1.17 pg/mL), IL-10 (DL: 2.88 pg/mL), IL-12p70 (DL: pg/mL), IL-13 (DL: 0.43 pg/mL), IL-17A (DL: 0.89 pg/mL), IL-22 (DL: 3.98 pg/mL), IL-23 (DL: 29.14 pg/mL), IL-27 (DL: 0.79 pg/mL), leptin (DL: 34.95 pg/mL), and tumour necrosis factor-α (DL: 3.10 pg/mL). IgA content was determined via radial immunodiffusion kit (DL: 8.5 mg/L; Kit IgA RID-ML, Binding Site Group, Birmingham, United Kingdom) following the manufacturer’s instructions. Values below the level of detection were reported as equal to it.