Synergy htx hybrid multi mode microplate reader

The splenocytes of each mouse groups were aseptically isolated using 70 μm cell strainer, washed and resuspended in ammonium chloride-Tris (ACT) buffer and deposited in 96-well plates at a concentration of 5.0 × 10⁶ cells/well with concanavalin A (ConA, 2 mg/mL). Splenocytes incubated with medium alone used as negative control. After cultivating Splenocytes and ConA for 96 h at 37 °C under atmosphere of 5% CO₂, 20 µL of MTS solution (Promega, Madison, WI, USA) was treated. And then, the plate was incubated for additional 2 h and read at 490 nm (OD₄₉₀) using a Synergy HTX Hybrid Multi Mode Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA). The proliferation of splenocyte were calculated by the stimulation index (SI): SI = OD₄₉₀ of stimulated cells/OD₄₉₀ of the negative control.

The concentration of hydrogen peroxide was determined by Amplex™ Red Hydrogen/HRP Peroxide Kit (cat.no A2218, Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Since the higher concentration of H₂O₂ able to be processed properly by this reagent is around 5 µM of H₂O₂, PAM with sodium pyruvate were diluted 10 and PAM without sodium pyruvate were diluted 10–100 times previously to the addition of the reagent. In this case, for hydrogen peroxide detection, 50 μL of the Amplex^TM Red/Horseradish Peroxidase (HRP) reagent were added to 50 μL of PAM in a dark 96-well plate and incubated for 30 min at room temperature. Subsequent fluorescence measurements were performed by means of a Synergy HTX Hybrid Multi Mode Microplate Reader (BioTek Instruments, Winooski, Vermont, USA), with fluorescence filters centred at λ_ex = 560/20 nm and λ_em = 590/20 nm as excitation and emission wavelengths, respectively. Concentrations of H₂O₂ in PAM generated by plasma treatment were obtained from fluorescence values using a calibration curve following the manufacturer’s protocol.

Determination of NO₂⁻ concentration in PAM was performed using Griess reagent^{78 (link)}. The Griess working solution used was obtained by dissolving 1% wt/v of sulphanilamide, 0.1% wt/v of NEED and 5% w/v of phosphoric acid in de-ionized water. 50 μL of Griess solution were added on 50 μL of sample in 96 well-plates. The plates were incubated for 10 min at room temperature protected from light. The absorbance was measured at λ_abs = 540 nm using a Synergy HTX Hybrid Multi Mode Microplate Reader (BioTek Instruments, Winooski, Vermont, USA). The [NO₂⁻] in each sample was determined from the absorbance values by using a calibration curve made from a commercial standard Sodium Nitrite 14 mM (CellBiolabs, cat.no 280201, San Diego, CA, USA) diluted on DMEM.